Tau Imaging Ligands and Their Uses in the Diagnosis and Treatment of Tauopathy

ABSTRACT

The present invention relates to antibody-based probes (including single domain antibody fragment, scFv molecules, antibodies, antibody fragments, diabodies, and the epitope-binding domains thereof) that are capable of immunospecifically and selectively binding to a phospho-serine-containing epitope of Tau, such as, for example, Tau-phospho-serine 396/404 peptide. Such imaging ligands are useful to detect pathological Tau protein conformer if present in a biological sample, especially in conjunction with the diagnosis of Alzheimer&#39;s disease or other tauopathy, and thus provide a diagnostic for Alzheimer&#39;s disease and other Tau pathologies. The scFv molecules of the present invention have utility as diagnostic markers for, Alzheimer&#39;s disease and related tauopathies and as pharmaceutical compositions for the treatment of such conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of, and claims priority to, U.S.patent application Ser. No. 16/151,555 (filed Oct. 4, 2018; pending),which a continuation of U.S. patent application Ser. No. 15/324,141(filed Jan. 5, 2017; issued as U.S. Pat. No. 10,132,818 on Nov. 20,2018), which is a § 371 Application of PCT/US2015/039205 (filed Jul. 6,2015, now lapsed), which application claims priority to U.S. ProvisionalPatent Appln. Ser. No. 62/021,897 (filed on Jul. 8, 2014; now lapsed),each of which application is hereby incorporated by reference herein inits entirety

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Nos.NS077239, AG032611 and AG020197 awarded by the National Institutes ofHealth (NIH). The government has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

This application includes one or more Sequence Listings pursuant to 37C.F.R. 1.821 et seq., which are disclosed in computer-readable media(file name: SIG-09-0927-WO-PCT Sequence Listing ST25.txt, created onJul. 3, 2015, and having a size of 28,918 bytes), which file is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to antibody-based probes (including singledomain antibody fragment, scFv molecules, antibodies, antibodyfragments, diabodies, and the epitope-binding domains thereof) that arecapable of immunospecifically and selectively binding to aphospho-serine-containing epitope of Tau, such as, for example,Tau-phospho-serine 396/404 peptide. Such imaging ligands are useful todetect pathological Tau protein conformer if present in a biologicalsample, especially in conjunction with the diagnosis of Alzheimer'sdisease or other tauopathy, and thus provide a diagnostic forAlzheimer's disease and other Tau pathologies. The scFv molecules of thepresent invention have utility as diagnostic markers for, Alzheimer'sdisease and related tauopathies and as pharmaceutical compositions forthe treatment of such conditions.

BACKGROUND OF THE INVENTION

Alzheimer's disease is the most common form of dementia affecting morethan 20 million people worldwide.

Diagnosis of the disease, in particular at an early point, istroublesome and difficult and there exists a need for accurate diagnosisof tauopathies such as Alzheimer's disease. Antibody detection ofabnormal Tau in cerebrospinal fluid has shown some promise (Blennow etal. “Cerebrospinal Fluid And Plasma Biomarkers In Alzheimer Disease,”Nat. Rev. Neurol. 6, 131-144 (2010) and Weiner et al. “The Alzheimer'sDisease Neuroimaging Initiative: A Review Of Papers Published Since ItsInception,” Alzheimers Dement. 9, e111-e194 (2013)).

Over the years, antibody detection of phospho-Tau protein incerebrospinal fluid has shown some utility for diagnosis of Alzheimer'sdisease (Blennow et al. “Cerebrospinal Fluid And Plasma Biomarkers InAlzheimer Disease,” Nat. Rev. Neurol. 6, 131-144 (2010); Lewis, J. etal. “Neurofibrillary Tangles, Amyotrophy And Progressive MotorDisturbance In Mice Expressing Mutant (P301L) Tau Protein,” Nat. Genet.25, 402-405; Weiner, M. W. et al. (2013) “The Alzheimer's DiseaseNeuroimaging Initiative: A Review Of Papers Published Since ItsInception,” Alzheimers Dement. 9: e111-e194), suggesting that furtherdevelopment in this arena is warranted (see, Congdon, E. E. (2014)“Harnessing The Immune System For Treatment And Detection Of TauPathology,” J. Alzheimers Dis. 40: S113-S121). However, CSF Tau levelsin other tauopathies are usually not altered compared to controls(Theunis, C. et al. “Efficacy And Safety Of A Liposome-Based VaccineAgainst Protein Tau, Assessed In Tau.P301L Mice That Model Tauopathy,”PLoS. One. 8, e72301 (2013); Hales, C. M. et al. (2013) “FromFrontotemporal Lobar Degeneration Pathology To Frontotemporal LobarDegeneration Biomarkers,” Int. Rev. Psychiatry 25:210-220), and imagingdyes may not detect pathological Tau in all tauopathies(Fodero-Tavoletti, M. T. et al. (2014) “Assessing THK523 Selectivity ForTau Deposits In Alzheimer's Disease And Non-Alzheimer's DiseaseTauopathies,” Alzheimers Res. Ther. 6:11). Imaging these Tau lesions inconcert with amyloid-β (Aβ) is more likely to lead to accurate diagnosisas the regional pattern of Tau aggregates differs between the differenttauopathies. Furthermore, all of them except Alzheimer's disease are inpart defined by lack of Aβ deposition. In vivo imaging of Aβ plaquesusing compounds that bind well to β-sheets is already in clinical use(Mason, N. S. et al. (2013) “Positron Emission Tomography RadioligandsFor In Vivo Imaging Of ABeta Plaques,” J. Labelled Comp. Radiopharm.56:89-95). Several such dye-based Tau-binding ligands have beenidentified recently in preclinical studies and some of those are beingevaluated (Fodero-Tavoletti, M. T. et al. (2014) “Assessing THK523Selectivity For Tau Deposits In Alzheimer's Disease And Non-Alzheimer'sDisease Tauopathies,” Alzheimers. Res. Ther. 6:11; Fodero-Tavoletti, M.T. et al. (2011) “18F-THK523: A Novel In Vivo Tau Imaging Ligand ForAlzheimer's Disease,” Brain 134:1089-1100; Zhang, W. et al. (2012) “AHighly Selective And Specific PET Tracer For Imaging Of TauPathologies,” J. Alzheimers. Dis. 31:601-612; Chien, D. T. et al. (2013)“Early Clinical PET Imaging Results With The Novel PHF-Tau Radioligand[F-18]-T807,” J. Alzheimers. Dis. 34:457-468; Maruyama, M. H. et al.(2013) “Imaging Of Tau Pathology In A Tauopathy Mouse Model And InAlzheimer Patients Compared To Normal Controls,” Neuron 79:1094-1108;Okamura, N. et al. (2005) “Quinoline And Benzimidazole Derivatives:Candidate Probes For In Vivo Imaging Of Tau Pathology In Alzheimer'sDisease,” J. Neurosci. 25:10857-10862; Harada, R., et al. (2013)“Comparison Of The Binding Characteristics Of [18F]THK-523 And OtherAmyloid Imaging Tracers To Alzheimer's Disease Pathology,” Eur. J. Nucl.Med. Mol. Imaging 40:125-132; Ono, M. et al. (2011) “Rhodanine AndThiohydantoin Derivatives For Detecting Tau Pathology In Alzheimer'sBrains,” ACS Chem. Neurosci. 2:269-275; Xia, C. F. et al. (2013)“[(18)F]T807, A Novel Tau Positron Emission Tomography Imaging Agent ForAlzheimer's Disease,” Alzheimers. Dement. 9:666-676; Chien, D. T. (2014)“Early Clinical PET Imaging Results With The Novel PHF-Tau Radioligand[F18]-T808,” J. Alzheimers. Dis. 38:171-184; Villemagne, V. L. et al.(2014) “In Vivo Evaluation Of A Novel Tau Imaging Tracer For Alzheimer'sDisease,” Eur. J. Nucl. Med. Mol. Imaging 41:816-826; Okamura, N. et al.(2014) “Non-Invasive Assessment Of Alzheimer's Disease NeurofibrillaryPathology Using 18F-THK5105 PET,” Brain 137:1762-1771). The hope andpromise for Tau based ligands is that they will be better than ligandsto monitor the status and progression of neurodegeneration.Antibody-based probes are likely to provide greater specificity fordetecting Tau lesions. In particular, smaller antibody fragments thatbind to Tau are attractive as ligands for in vivo imaging to detect Taulesions in patients with Alzheimer's disease or other tauopathies.

Within the cancer field, therapeutic antibodies have routinely beenco-developed as imaging agents, and several such antibodies and Fabmolecules are FDA approved for tumor imaging (Kaur, S. et al. “RecentTrends In Antibody-Based Oncologic Imaging,” Cancer Lett. 315, 97-111(2012)).

The present inventors have found antibody-derived imaging ligands thatprovide excellent specificity for detecting Tau lesions, and inparticular smaller single-chain variable antibody fragments (scFvmolecules) which are attractive for in vivo imaging of Tau aggregates.It is envisaged that these antibody-derived imaging ligands can beuseful in monitoring disease progression of Tau pathology, the efficacyof Tau-targeting therapies, and to identify Aβ negative tauopathies.

SUMMARY OF THE INVENTION

The present invention relates to antibody-based probes (including singledomain antibody fragment, scFv molecules, antibodies, antibodyfragments, diabodies, and the epitope-binding domains thereof) that arecapable of immunospecifically and selectively binding to aphospho-serine-containing epitope of Tau, such as, for example,Tau-phospho-serine 396/404 peptide. Such imaging ligands are useful todetect pathological Tau protein conformer if present in a biologicalsample, especially in conjunction with the diagnosis of Alzheimer'sdisease or other tauopathy, and thus provide a diagnostic forAlzheimer's disease and other Tau pathologies. The scFv molecules of thepresent invention have utility as diagnostic markers for, Alzheimer'sdisease and related tauopathies and as pharmaceutical compositions forthe treatment of such conditions.

In detail, the invention concerns a binding molecule that is capable ofimmunospecifically binding to a phosphorylated Tau peptide having anamino acid sequence consisting of the amino acid sequence of the Tau396/404 peptide (SEQ ID NO:7): TDHGAEIVYKSPVVSGDTSPRHL, wherein theserine residues at positions 11 and 19 thereof (shown underlined) arephosphorylated, wherein the binding molecule is additionally capable ofimmunospecifically binding to phosphorylated Tau with greaterselectivity than to non-phosphorylated Tau.

The invention also pertains to the embodiment of such a binding moleculewherein the molecule is an antibody or comprises an epitope-bindingfragment thereof. The invention further pertains to the embodiment ofany of such binding molecules wherein the binding molecule comprisessuch an epitope-binding fragment, and more particularly wherein themolecule is an isolated CDR, a single domain antibody fragment, an scFvor a diabody. The invention particularly pertains to the embodiment ofany of such binding molecules wherein upon peripheral injection into arecipient, the binding molecule substantially co-localizes with a Tauaggregate.

The invention also pertains to the embodiment of such binding moleculeswherein the epitope-binding fragment comprises any one, any two, anythree, any four, any five or all six of:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:12;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:13;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:14;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:15;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:16;and/or(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:17.

The invention also pertains to the embodiment of such binding moleculeswherein the binding molecule is scFv235 (SEQ ID NO:18).

The invention also pertains to the embodiment of any of such bindingmolecules which is detectably labeled, and especially, any of suchbinding molecules wherein the detectable label is a fluorescent label, achemoluminescent label, a paramagnetic label, a radioisotopic label oran enzyme label

The invention also pertains to the use of any of the above-describedbinding molecules for detecting or measuring the presence or amount ofthe phosphorylated Tau protein in the brain, or in a biological fluid(e.g., cerebrospinal fluid, blood, serum, plasma, etc.) of a recipientsubject. The invention particularly pertains to such a use, the usewherein the detection or measurement comprises in vivo or ex vivoimaging of the binding molecule bound to the phosphorylated Tau protein,and more particularly, wherein the detection or measurement is fordiagnosing Alzheimer's disease or another tauopathy of a subject.

The invention also pertains to an in vivo medicament for the treatmentof Alzheimer's disease or another tauopathy of a subject, wherein themedicament comprises the binding molecule of any of claims 1-8 in anamount effective to treat the Alzheimer's disease or other tauopathy,and one or more carriers, diluents and/or stabilizers.

The invention also pertains to the use of such an in vivo medicament forthe treatment of Alzheimer's disease or another tauopathy of thesubject.

The invention particularly pertains to the above-recited uses, whereinthe subject is a human.

The invention also pertains to a kit for detecting or measuring thepresence or amount of the phosphorylated Tau protein in the brain of asubject, or for diagnosing Alzheimer's disease or another tauopathy in asubject, wherein the kit comprises any of the above-recited bindingmolecules.

The invention further pertains to any of the above-recited uses, or toany of the above-recited medicament, or to any of the above-recitedkits, wherein the tauopathy is selected from the group comprisingfrontotemporal dementia, parkinsonism linked to chromosome 17 (FTDP-17),progressive supranuclear palsy, corticobasal degeneration, Pick'sdisease, progressive subcortical gliosis, tangle only dementia, diffuseneurofibrillary tangles with calcification, argyrophilic grain dementia,amyotrophic lateral sclerosis parkinsonism-dementia complex, dementiapugilistica, Down syndrome, Gerstmann-Straussler-Scheinker disease,Hallerworden-Spatz disease, inclusion body myositis, Creutzfeld-Jakobdisease, multiple system atropy, Niemann-Pick disease type C, prionprotein cerebral amyloid angiopathy, subacute sclerosingpanencephalitis, myotonic dystrophy, non-guanamian motor neuron diseasewith neurofibrillary tangles, postencephalitic parkinsonism, acutetraumatic brain injury and chronic traumatic encephalopathy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show the binding of randomly selected scFv clones generatedfrom antibody 6B2G12 (FIGS. 1A and 1B) to the non-phosphorylated epitopeof SEQ ID NO:27 or the phosphorylated Tau epitope of SEQ ID NO:28. Theclones of FIG. 1A were panned against the phosphorylated epitope (SEQ IDNO:28). The clones of FIG. 1B were panned against the non-phosphorylatedepitope (SEQ ID NO:27).

FIGS. 2A-2E: scFv235 binds selectively to phospho-Tau epitope and to Tauprotein in Alzheimer's brain homogenate.

FIG. 2A shows that scFv235 selectively binds to phospho-Tau-serine396/404 (P-Ser396/404) epitope in ELISA compared to its non-phosphoequivalent epitope (Ser396/404).

FIG. 2B shows that scFv235 specifically binds to Tau of Alzheimer'sdisease brain homogenate. Incubation of a brain homogenate from anindividual with Alzheimer's disease with scFv235 results in theimmunoprecipitation of Tau bands in the range of 50-70 kDa. Westernblots with CP27 (human Tau) and polyclonal total Tau antibody (Dako)showed similar results with no other bands detected. For comparison, alow speed supernatant (LSS) of the brain homogenate is shown in anadjacent lane from the same blot with several additional Tau positivebands.

FIG. 2C: Incubation of a brain homogenate from an individual withAlzheimer's disease with 6B2G12 results in the immunoprecipitation ofTau bands in the range of 50-70 kDa as well as degradation fragments inthe range of 20-30 kDa. Western blots with CP27 (human Tau) andpolyclonal total Tau antibody (Dako) showed greater detection of intactTau with CP27 and smaller Tau fragments with Dako Tau. For comparison, alow speed supernatant (LSS) of the brain homogenate is shown in anadjacent lane from the same blot with several additional Tau positivebands.

FIG. 2D: Shows the results of differential staining for four human brainsections. Row 1: brain section from Alzheimer's disease individual AD-1;Row 2: brain section from Alzheimer's disease individual AD-2; Row 3:brain section from Pick's disease individual; Row 4: brain section fromhealthy control individual. Column A: section stained with Hoechst toshow nuclei; Column B: section stained with scFv235; Column C: sectionstained with PHF1; Column D: sections merged to show localization (seearrows). Staining of human brain sections with scFv235 revealedextensive neuronal staining that showed partial localization with PHF1staining. Very limited staining was observed in control human tissue.Scale bar=20 μm.

FIG. 2E: Shows that scFv 235 binds to pathological Tau on Alzheimer'sdisease human brain sections, but exhibits limited binding to controlhuman brain sections.

FIGS. 3A-3D: In vivo imaging of Tau inclusions in P301L, htau/PS1, htau,Tg-SwDI and wild-type mice after intracarotid or intravenously injectedfluorescently labeled scFv235 or 6B2G12. Representative images fromvarious groups are shown in different panels.

FIG. 3A: Intracarotid Imaging. Images recorded prior to injection(Column A) and at specified intervals (Column B, Column C and Column D)post-injection. Row 1: images of P301L (12 months old) transgenic mouse,injected with scFv235 tagged with near infrared dye (680 nm) and imagedbefore injection (Column A), and at 38 minutes (Column B), 82 minutes(Column C) and 220 minutes (Column D) post-injection. Signal is detectedthroughout the body with highest intensity in the brain that slowlydecreases over time. Brain signal peaked at 35-38 min (1714% abovepre-injection baseline signal), and remained strong at 82 and 330minutes post-injection (1675% and 1468%, respectively). Row 2: imagesfrom an scFv235-injected htau/PS1 (22 months) transgenic mouse, recordedbefore injection, and at 37 minutes (Column B), 65 minutes (Column C)and 176 minutes (Column D) post-injection. Again, the most intensesignal was detected in the brain and it gradually decreased over time.Brain signal peaked at 37 minutes (1443% above pre-injection baselinesignal), and remained strong at 65 and 176 minutes post-injection (1144%and 1093%, respectively). Row 3: images of scFv235-injected wt (9months) mouse, before injection and at 37 minutes (Column B), 75 minutes(Column C) and 265 minutes (Column D) post-injection. Very limited brainsignal was detected post-injection with no signal in periphery. Row 4:images of 6B2G12-injected P301L (10 months) transgenic mouse, recordedbefore injection and at 37 minutes (Column B), 170 minutes (Column C)and 291 minutes (Column D) post-injection. Signal is strongest in thebrain and gradually decreases over time, with similar signal obtainedfrom 20-95 minutes post-injection (600-632% above baseline value), witha modest reduction at the depicted 170 and 291 minutes (497% and 431%,respectively). Row 5: images of 6B2G12-injected wt mouse (8 months),recorded before injection and at 37 minutes (Column B), 90 minutes(Column C) and 225 minutes (Column D) post-injection. Virtually nosignal is detected. The scale bar shows maximum pixel intensity, whereasthe region of interest (ROI) is total radiant efficiency (TRE) of summedpixel intensity.

FIG. 3B: Quantitative analysis of IVIS (in vivo Imaging System) brainsignal over time after intracarotid injection: Highest signal wasdetected in P301L mice injected with scFv235. The older mice (11 and 12months) had total radiant efficiency (TRE) peak at 2.23E+11 and 2.64E+11respectively, whereas the younger mice (3 and 8 months) had lower peaksignals (1.88E+11 and 1.86E+11 respectively). The same P301L model (7-10months) had strong but lesser brain signal after 6B2G12 injection,ranging from 2.20E+11 to 1.16E+11. Similar signal intensity was observedin an old scFv235-injected htau/PS1 mouse (22 months; peak at 1.40E+11)but limited in a 7 months old htau/PS1 mouse, which was confirmed tolack Tau pathology. Wt mice (12-13 months) and one htau mouse (13months) had low signal at all time points and had no Tau pathology.Injection of the fluorescent-tag alone in an old htau/PS1 mouse (23months) and a P301L mouse (7 months) gave a higher brain signal than inwt mice, injected with scFv235 or antibody, but it was substantiallyless than in any of the tauopathy mice injected with scFv235 or 6B2G12.These two dye injected mice were confirmed to have extensive Taupathology (see FIG. 4B).

FIG. 3C: Intravenous Imaging. Images recorded prior to injection (ColumnA) and at specified intervals (Column B, Column C and Column D)post-injection. Row 1: images of P301L (13 months old) transgenic mouse,injected with scFv235 tagged with near infrared dye (680 nm) and imagedbefore injection (Column A), and at 18 minutes (Column B), 210 minutes(Column C) and 11520 minutes (day 8) (Column D) post-injection. Peakbrain signal was detected at 18 min (1754% above pre-injectionbaseline), with lesser signal at 210 min (18% reduction from peaksignal), that had substantially subsided at 8 days (43% reduction). Row2: images from an Tg-SwDI Aβ plaque mouse (12 months) recorded beforeinjection, and at 25 minutes (Column B), 60 minutes (Column C) and 11520minutes (day 8) (Column D) post-injection. Very limited brain signal wasdetected post-injection with no signal in periphery. Row 3: images of6B2G12-injected P301L (7 months) transgenic mouse, before injection andat 25 minutes (Column B), 120 minutes (Column C) and 11520 minutes (day8) (Column D) post-injection. Brain signal was strong at 25 minutes(1211% above pre-injection baseline), peaked at 35 minutes (1445%), withlesser signal at 120 minutes (11% reduction from peak signal) and wasmuch weaker by 8 and 12 days (67% and 70% reduction, respectively). Row4: images of 6B2G12 injected Tg-SwDI mouse (12 months), recorded beforeinjection and at 25 minutes (Column B), 180 minutes (Column C) and 11520minutes (day 8) (Column D) post-injection. Very limited brain signal wasdetected post-injection with no signal in periphery.

FIG. 3D: Quantitative analysis of IVIS brain signal over time afterintravenous injection: scFv235 and 6B2G12 were injected into P301L mice.These mice could be imaged at earlier time points post-injection thanthe intracarotid injected mice as the latter route requires longerpostoperative care to prevent bleeding. Peak signal was generallyobtained within the first hour post-injection, depending on the animal,at similar intervals for all probes. Overall signal was substantiallyhigher for scFv235 and 6B2G12 compared to control IgG, even though theIgG injected mice had the most robust Tau pathology (see Table 6). One6B2G12 injected mouse (B15) had comparable IVIS signal to the IgGinjected mice but more modest Tau pathology. The signal graduallysubsided over 14 days at different rates for individual P301L mice. Verylimited signal was detected at all time points in injected Tg-SwDI or wtmice with either probe.

FIGS. 4A-4D: Co-localization of injected scFv235 and 6B2G12 withintraneuronal Tau protein, markers of endosomes-autophagosomes-lysosomesand microglia. Brains were removed 3-4 hours following intracarotidinjection and IVIS imaging, fixed, sectioned coronally and stained for:(1) Tau with Tau5 (total Tau), MC1 (conformational), PHF1 (phospho-Tau),and (2) early endosomes (EEA1); (3) late endosomes/lysosomes (Rab 7);and (4) autophagosomes (LC3, P62). Sections were also stained withmicroglia (Iba-1).

FIG. 4A: scFv235-injected P301L mice (Rows 1-2, 4 and 7: mouse “A12;”Row 3: mouse “BBB;” Rows 5-6: mouse “A13”) were imaged to identifynuclei (Column A), imaged with scFV235 (Column B), imaged to show PHF1(Column C), and a merged image was created (Column D). Images showedpartial co-localization with MC1, Tau5, and PHF1, and completeco-localization with Rab7, EEA1, LC3 and P62.

FIG. 4B: scFv235-injected htau/PS1 mice (Rows 1, 5, 7-8: mouse “A47;”Rows 2-4 and 6: mouse “B32”) were imaged to identify nuclei (Column A),imaged with scFV235 (Column B), imaged to show PHF1 (Column C), and amerged image was created (Column D). Images showed partialco-localization of the injected scFv235 with MC1, Tau5, and Iba-1 (notshown) and complete co-localization with PHF1, Rab7, EEA1, LC3 and P62antibodies.

FIG. 4C: 6B2G12-injected P301L mice (Rows 1 and 7: mouse “A52;” Rows2-6: mouse “A50”) were imaged to identify nuclei (Column A), imaged withscFV235 (Column B), imaged to show PHF1 (Column C), and a merged imagewas created (Column D).

Images showed partial co-localization with MC1, Tau5, PHF1, Rab7, EEA1,LC3 and P62.

FIG. 4D: Wt mice injected with scFv235 (Rows 1-3) or 6B2G12 (Rows 4-6)were imaged to identify nuclei (Column A), imaged with scFV235 (ColumnB), imaged to show PHF1 (Column C), and a merged image was created(Column D). Images showed limited signal from the antibody fragment andlimited staining with the antibody markers except for normal Taudetected with Tau5. Nuclei are stained blue with Hoechst nuclear stain.Scale bar=10 μm. Arrows point at some of the neurons with partialco-localization. No arrows were used when there was completeco-localization.

FIG. 5 shows that scFv235 co-localized with pathological Tau withinbrain neurons following intracarotid injection in P301L (F6) andhtau/PS1 (A47) tangle mice, but did not have detectable uptake incontrol wild-type mice (R1).

FIGS. 6A-6F show the excellent correlation that was observed of IVISsignal with (FIGS. 6A-6B) brain tissue probe signal (scFv235: r=0.98,6B2G12: r=0.87) and with (FIGS. 6C-6D) Tau pathology (scFv235: r=0.94,6B2G12: r=0.75). The brain tissue probe signal correlated as well nicelywith (FIGS. 6E-6F) brain Tau pathology (scFv235: r=0.99, 6B2G12:r=0.97).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to antibody-based probes (including singledomain antibody fragment, scFv molecules, antibodies, antibodyfragments, diabodies, and the epitope-binding domains thereof) that arecapable of immunospecifically and selectively binding to aphospho-serine-containing epitope of Tau, such as, for example,Tau-phospho-serine 396/404 peptide. Such imaging ligands are useful todetect pathological Tau protein conformer if present in a biologicalsample, especially in conjunction with the diagnosis of Alzheimer'sdisease or other tauopathy, and thus provide a diagnostic forAlzheimer's disease and other Tau pathologies. The scFv molecules of thepresent invention have utility as diagnostic markers for, Alzheimer'sdisease and related tauopathies and as pharmaceutical compositions forthe treatment of such conditions.

The antibody-based probes of the present invention provide greaterspecificity than β-sheet dyes for detecting Tau lesions in patients withAD or other tauopathies. In particular, smaller antibody fragments thatbind to Tau are attractive as ligands for in vivo imaging. Their smallersize compared to antibodies leads to better access to Tau aggregates.Another advantage is their relatively rapid clearance from thecirculation compared to unmodified antibodies that have longerhalf-lives. Within the cancer field, therapeutic antibodies haveroutinely been co-developed as imaging agents, and several suchantibodies and Fab's or smaller diabodies and scFv molecules with betterpharmacokinetic properties approved or proposed as tumor imaging agents(see, Kaur, S. et al. (2012) “Recent Trends In Antibody-Based OncologicImaging,” Cancer Lett. 315:97-111; Olafsen, T. et al. (2010) “AntibodyVectors For Imaging,” Semin. Nucl. Med. 40:167-181).

I. Tau and the Preferred Immunogenic Tau Peptides of the PresentInvention

As used herein, the term “Tau” is synonym with the Tau protein andrefers to any of the Tau protein isoforms (identified in for exampleUniProt as P10636, 1-9). The amino acid numbering of Tau is given withrespect to SEQ ID NO: 1 as shown below, Met being amino acid 1. “P-Tau”refers to a Tau protein that has been phosphorylated at one or moreserine or threonine residues. For example P-Ser 396/404 refers to apolypeptide of Tau that comprises the amino acid sequence of SEQ ID NO:1wherein serine residues 396 and 404 are phosphorylated:

SEQ ID NO: 1: MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTDAGLKESPLQT PTEDGSEEPG SETSDAKSTP TAEDVTAPLVDEGAPGKQAA AQPHTEIPEG TTAEEAGIGD TPSLEDEAAGHVTQARMVSK SKDGTGSDDK KAKGADGKTK IATPRGAAPPGQKGQANATR IPAKTPPAPK TPPSSGEPPK SGDRSGYSSPGSPGTPGSRS RTPSLPTPPT REPKKVAVVR TPPKSPSSAKSRLQTAPVPM PDLKNVKSKI GSTENLKHQP GGGKVQIINKKLDLSNVQSK CGSKDNIKHV PGGGSVQIVY KPVDLSKVTSKCGSLGNIHH KPGGGQVEVK SEKLDFKDRV QSKIGSLDNITHVPGGGNKK IETHKLTFRE NAKAKTDHGA EIVYKSPVVSGDTSPRHLSN VSSTGSIDMV DSPQLATLAD EVSASLAKQG L

Tau is a soluble microtubule-associated protein that is dynamicallyphosphorylated and dephosphorylated by a host of kinase enzymes duringthe cell cycle. Tau's ability to stabilize microtubules is dependent onthe extent of its phosphorylation. In its dephosphorylated form, theprotein is able to interact with tubulin to stabilize microtubules andpromote tubulin assembly into microtubules (which form the cytoskeletonof the cell and are the major constituents of the mitotic spindles thatpull apart eukaryotic chromosomes in mitosis). In its phosphorylatedform, Tau is able to dissociate from microtubules, thereby permittingmitosis to occur. The phosphorylation of Tau acts thus as a directmicrotubule association-dissociation switch within the neuron (Pedersen,J. T. et al. (2015) “Tau Immunotherapy For Alzheimer's Disease,” TrendsMol. Med. 2015 Apr. 3. pii: S1471-4914(15)00058-1; pages 1-9, herebyincorporated by reference herein in its entirety).

Hyperphosphorylation of Tau can result in the formation of insolubleself-assembling “tangles,” referred to herein as “Tau aggregates,” ofpaired helical filaments and straight filaments. Such Tau aggregates maybe intracellular (e.g., intraneuronal), but may also form outside of thecells. The presence of Tau aggregates impairs Tau's ability to stabilizemicrotubules and thus leads to microtubule disassembly, dendritic spinalcollapse, and the degeneration of axons. Normal Tau contains, on averagetwo phosphorylated sites; the hyperphosphorylated Tau filaments averageseven to eight phosphorylated sites. Hyperphosphorylated Tau is the mainconstituent of the intracellular neurofibrillary tangles that are a mainhallmark of Alzheimer's Disease.

II. The Preferred Antibodies and Epitope-Binding Fragments of thePresent Invention

As used herein, the term “antibody” refers to an intact immunoglobulinas well as a molecule having an epitope-binding fragment thereof.Naturally occurring antibodies typically comprise a tetramer which isusually composed of at least two heavy (H) chains and at least two light(L) chains. Each heavy chain is comprised of a heavy chain variableregion (abbreviated herein as VH) and a heavy chain constant region,usually comprised of three domains (CH1, CH2 and CH3). Heavy chains canbe of any isotype, including IgG (IgG1, IgG2, IgG3 and IgG4 subtypes),IgA (IgA1 and IgA2 subtypes), IgM and IgE. Each light chain is comprisedof a light chain variable region (abbreviated herein as VL) and a lightchain constant region (CL). Light chains include kappa chains and lambdachains. The heavy and light chain variable region is typicallyresponsible for antigen recognition, while the heavy and light chainconstant region may mediate the binding of the immunoglobulin to hosttissues or factors, including various cells of the immune system (e.g.,effector cells) and the first component (C1q) of the classicalcomplement system. The VH and VL regions can be further subdivided intoregions of hypervariability, termed “complementarity determiningregions,” that are interspersed with regions of more conserved sequence,termed “framework regions” (FR). Each VH and VL is composed of three CDRDomains and four FR Domains arranged from amino-terminus tocarboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.The variable regions of the heavy and light chains contain a bindingdomain that interacts with an antigen. Of particular relevance areantibodies and their epitope-binding fragments that have been “isolated”so as to exist in a physical milieu distinct from that in which it mayoccur in nature or that have been modified so as to differ from anaturally occurring antibody in amino acid sequence.

Fragments of antibodies (including Fab and (Fab)₂ fragments) thatexhibit epitope-binding ability can be obtained, for example, byprotease cleavage of intact antibodies. More preferably, such fragmentswill be single domain antibody fragments, scFv molecules, and theepitope-binding domains of antibodies, etc., that are formed usingrecombinant techniques. For example, although the two domains of the Fvfragment, VL and VH, are encoded by separate genes, such gene sequencesor their encoding cDNA can be joined, using recombinant methods, by aflexible linker (typically of about 10, 12, 15 or more amino acidresidues) that enables them to be made as a single protein chain inwhich the VL and VH regions associate to form monovalent epitope-bindingmolecules (known as single-chain Fv (scFv) molecules; see e.g., Bird etal., (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. (U.S.A.) 85:5879-5883). Alternatively, by employing aflexible linker that is too short (e.g., less than about 9 residues) toenable the VL and VH regions of a single polypeptide chain to associatetogether, one can form a bispecific antibody, diabody, or similarmolecule (in which two such polypeptide chains associate together toform a bivalent epitope-binding molecule) (see for instance PNAS USA90(14), 6444-8 (1993) for a description of diabodies). Single domainantibody fragments possess only one variable domains (e.g., VL or VH).Examples of the epitope-binding fragments encompassed within the presentinvention include (i) Fab′ or Fab fragments, a monovalent fragmentconsisting of the VL, VN, CL and CH1 domains, or a monovalent antibodyas described in WO2007059782; (ii) F(ab′)₂ fragments, bivalent fragmentscomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) Fd fragments consisting essentially of the VH and CH1domains; (iv) Fv fragments consisting essentially of a VL and VH domain,(v) dAb fragments (Ward et al., Nature 341, 544-546 (1989)), whichconsist essentially of a VH domain and also called domain antibodies(Holt et al; Trends Biotechnol. 2003 November; 2i(11):484-90); (vi)camelid or nanobodies (Revets et al.; Expert Opin Biol Ther. 2005January; 5_(1):111-124) and (vii) isolated complementarity determiningregions (CDR). Furthermore, although the two domains of the Fv fragment,VL and VH, are coded for by separate genes, they may be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain antibodies or single chainFv (scFv), see for instance Bird et al., Science 242, 423-426 (1988) andHuston et al., PNAS USA 85, 5879-5883 (1988)). These and other usefulantibody fragments in the context of the present invention are discussedfurther herein. It also should be understood that the term antibody,unless specified otherwise, also includes antibody-like polypeptides,such as chimeric antibodies and humanized antibodies, and antibodyfragments retaining the ability to specifically bind to the antigen(antigen-binding fragments) provided by any known technique, such asenzymatic cleavage, peptide synthesis, and recombinant techniques. Anantibody as generated can possess any isotype. As used herein, “isotype”refers to the immunoglobulin class (for instance IgG1, IgG2, IgG3, IgG4,IgD, IgA, IgE, or IgM) that is encoded by heavy chain constant regiongenes. The choice of isotype typically will be guided by the desiredeffector functions, such as ADCC induction. Exemplary isotypes are IgG1,IgG2, IgG3, and IgG4. Either of the human light chain constant regions,kappa or lambda, may be used. If desired, the class of an anti-Tauantibody of the present invention may be switched by known methods. Forexample, an antibody of the present invention that was originally IgMmay be class switched to an IgG antibody of the present invention.Further, class switching techniques may be used to convert one IgGsubclass to another, for instance from IgG1 to IgG2. Thus, the effectorfunction of the antibodies of the present invention may be changed byisotype switching to, e.g., an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, orIgM antibody for various therapeutic uses. In one embodiment an antibodyof the present invention is an IgG1 antibody, for instance an IgG1, κ.

Such antibody fragments are obtained using conventional techniques knownto those of skill in the art. For example, F(ab′)2 fragments may begenerated by treating antibody with pepsin. The resulting F(ab′)2fragment may be treated to reduce disulfide bridges to produce Fab′fragments. Fab fragments may be obtained by treating an IgG antibodywith papain; Fab′ fragments may be obtained with pepsin digestion of IgGantibody. An F(ab′) fragment may also be produced by binding Fab′described below via a thioether bond or a disulfide bond. A Fab′fragment is an antibody fragment obtained by cutting a disulfide bond ofthe hinge region of the F(ab′)2. A Fab′ fragment may be obtained bytreating an F(ab′)2 fragment with a reducing agent, such asdithiothreitol. Antibody fragment may also be generated by expression ofnucleic acids encoding such fragments in recombinant cells (see forinstance Evans et al., J. Immunol. Meth. 184, 123-38 (1995)). Forexample, a chimeric gene encoding a portion of an F(ab′)2 fragment couldinclude DNA sequences encoding the CH1 domain and hinge region of the Hchain, followed by a translational stop codon to yield such a truncatedantibody fragment molecule. Suitable fragments capable of binding to adesired epitope may be readily screened for utility in the same manneras an intact antibody.

In one embodiment, such antibody fragments are a monovalent antibody,preferably a monovalent antibody as described in PCT Publication WO2007/059782 (which is incorporated herein by reference in its entirety)having a deletion of the hinge region. Such an antibody may beconstructed by a method comprising: i) providing a nucleic acidconstruct encoding the light chain of said monovalent antibody, saidconstruct comprising a nucleotide sequence encoding the VL region of aselected antigen specific anti alpha-synuclein antibody and a nucleotidesequence encoding the constant CL region of an Ig, wherein saidnucleotide sequence encoding the VL region of a selected antigenspecific antibody and said nucleotide sequence encoding the CL region ofan Ig are operably linked together, and wherein, in case of an IgG1subtype, the nucleotide sequence encoding the CL region has beenmodified such that the CL region does not contain any amino acidscapable of forming disulfide bonds or covalent bonds with other peptidescomprising an identical amino acid sequence of the CL region in thepresence of polyclonal human IgG or when administered to an animal orhuman being; ii) providing a nucleic acid construct encoding the heavychain of said monovalent antibody, said construct comprising anucleotide sequence encoding the VH region of a selected antigenspecific antibody and a nucleotide sequence encoding a constant CHregion of a human Ig, wherein the nucleotide sequence encoding the CHregion has been modified such that the region corresponding to the hingeregion and, as required by the Ig subtype, other regions of the CHregion, such as the CH3 region, does not comprise any amino acidresidues which participate in the formation of disulfide bonds orcovalent or stable non-covalent inter-heavy chain bonds with otherpeptides comprising an identical amino acid sequence of the CH region ofthe human Ig in the presence of polyclonal human IgG or whenadministered to an animal human being, wherein said nucleotide sequenceencoding the VH region of a selected antigen specific antibody and saidnucleotide sequence encoding the CH region of said Ig are operablylinked together; iii) providing a cell expression system for producingsaid monovalent antibody; iv) producing said monovalent antibody byco-expressing the nucleic acid constructs of (i) and (ii) in cells ofthe cell expression system of (iii).

Similarly, in one embodiment, the antibody is a monovalent antibody,which comprises:

-   (i) a variable region of an antibody of the invention as described    herein or an antigen binding part of the said region, and-   (ii) a CH region of an immunoglobulin or a fragment thereof    comprising the CH2 and CH3 regions, wherein the CH region or    fragment thereof has been modified such that the region    corresponding to the hinge region and, if the immunoglobulin is not    an IgG4 subtype, other regions of the CH region, such as the CH3    region, do not comprise any amino acid residues, which are capable    of forming disulfide bonds with an identical CH region or other    covalent or stable non-covalent inter-heavy chain bonds with an    identical CH region in the presence of polyclonal human IgG.

In a further embodiment, the heavy chain of the monovalent antibody hasbeen modified such that the entire hinge has been deleted.

In another further embodiment, the sequence of said monovalent antibodyhas been modified so that it does not comprise any acceptor sites forN-linked glycosylation.

As used herein, an antibody or an epitope-binding fragment thereof issaid to “immunospecifically” bind a region of another molecule (i.e., anepitope) if it reacts or associates more frequently, more rapidly, withgreater duration and/or with greater affinity or avidity with thatepitope relative to alternative epitopes. It is also understood byreading this definition that, for example, an antibody or anepitope-binding fragment thereof that specifically binds to a firsttarget may or may not specifically or preferentially bind to a secondtarget.

As used herein, the term “binding” in the context of the binding of anantibody or binding fragment thereof to a predetermined antigentypically refers to binding with an affinity corresponding to a K_(D) ofabout 10⁻⁷ M or less, such as about 10⁻⁸ M or less, such as about 10⁻⁹ Mor less when determined by, for instance, surface plasmon resonance(SPR) technology in a BIAcore 3000 instrument (preferably using theantibody as the ligand and the antigen as the analyte), and which bindsto the predetermined antigen with an affinity corresponding to a K_(D)that is at least ten-fold lower, such as at least 100 fold lower, forinstance at least 1,000 fold lower, such as at least 10,000 fold lower,for instance at least 100,000 fold lower than its affinity for bindingto a non-specific antigen (e.g., BSA, casein) other than thepredetermined antigen or a closely-related antigen. The amount withwhich the affinity is lower is dependent on the K_(D) of the antibody,so that when the K_(D) of the antibody is very low (that is, theantibody is highly specific), then the amount with which the affinityfor the antigen is lower than the affinity for a non-specific antigenmay be at least 10,000 fold. The term “k_(d)” (sec⁻¹ or 1/s), as usedherein, refers to the dissociation rate constant of a particularantibody-antigen interaction. Said value is also referred to as thek_(off) value. The term “k_(a)” (M⁻¹×sec⁻¹ or 1/M), as used herein,refers to the association rate constant of a particular antibody-antigeninteraction. The term “K_(D)” (M), as used herein, refers to thedissociation equilibrium constant of a particular antibody-antigeninteraction. The term “K_(A)” (M⁻¹ or 1/M), as used herein, refers tothe association equilibrium constant of a particular antibody-antigeninteraction and is obtained by dividing the k_(a) by the k_(a).

As used herein, an antibody or an epitope-binding fragment thereof issaid to “selectively” bind to a phosphorylated peptide epitope if itimmunospecifically binds to such epitope with higher affinity than itbinds (if it binds at all) to a non-phosphorylated peptide epitopehaving the same amino acid sequence. Most preferably, such higheraffinity will be at least 10-fold higher, at least 30-fold higher, atleast 100-fold higher, at least 300-fold higher, at least 1,000-foldhigher, at least 3,000-fold higher, or at least 10,000-fold higher.scFv235 “selectively” binds to the phosphorylated P-Ser396/404 peptiderelative to the non-phosphorylated peptide (exhibiting a “selectivity”of about 4,000-fold). The extent of “selectivity” of an antibody, or ofan epitope-binding fragment thereof, for phosphorylated Tau isdetermined by comparing, via ELISA or Biacore, the affinity with whichan scFv of such antibody immunospecifically binds to anon-phosphorylated target Tau peptide and to a phosphorylated variantthereof.

The term “epitope” means an antigenic determinant capable of specificbinding to an antibody. Epitopes usually consist of surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. Conformational and non-conformationalepitopes are distinguished in that the binding to the former, but notthe latter, is lost in the presence of denaturing solvents. The epitopemay comprise amino acid residues directly involved in the binding (alsocalled immunodominant component of the epitope) and other amino acidresidues, which are not directly involved in the binding, such as aminoacid residues which are effectively blocked by the specifically antigenbinding peptide (in other words, the amino acid residue is within thefootprint of the specifically antigen binding peptide).

As used herein, the term “epitope-binding fragment of an antibody” meansa fragment of an antibody capable of immunospecifically binding to anepitope. An epitope-binding fragment may contain 1, 2, 3, 4, 5 or all 6of the CDR Domains of such antibody and, although capable ofimmunospecifically binding to such epitope, may exhibit animmunospecificity, affinity or selectivity toward such epitope thatdiffers from that of such antibody. Preferably, however, anepitope-binding fragment will contain all 6 of the CDR Domains of suchantibody. An epitope-binding fragment of an antibody may be a singlepolypeptide chain (e.g., an scFv), or may comprise two or morepolypeptide chains, each having an amino-terminus and a carboxylterminus (e.g., a diabody, an Fab fragment, an Fab₂ fragment, etc.).

The antibodies of the present invention, and their Tau epitope-bindingfragments will preferably be “humanized,” particularly if employed fortherapeutic purposes. The term “humanized” refer to a chimeric molecule,generally prepared using recombinant techniques, having an antigenbinding site derived from an immunoglobulin from a non-human species anda remaining immunoglobulin structure based upon the structure and/orsequence of a human immunoglobulin. The antigen-binding site maycomprise either complete non-human antibody variable domains fused tohuman constant domains, or only the complementarity determining regions(CDRs) of such variable domains grafted to appropriate human frameworkregions of human variable domains. The framework residues of suchhumanized molecules may be wild type (e.g., fully human) or they may bemodified to contain one or more amino acid substitutions not found inthe human antibody whose sequence has served as the basis forhumanization. Humanization lessens or eliminates the likelihood that aconstant region of the molecule will act as an immunogen in humanindividuals, but the possibility of an immune response to the foreignvariable region remains (LoBuglio, A. F. et al. (1989) “Mouse/HumanChimeric Monoclonal Antibody In Man: Kinetics And Immune Response,”Proc. Natl. Acad. Sci. (U.S.A.) 86:4220-4224). Another approach focusesnot only on providing human-derived constant regions, but modifying thevariable regions as well so as to reshape them as closely as possible tohuman form. It is known that the variable regions of both heavy andlight chains contain three complementarity-determining regions (CDRs)which vary in response to the antigens in question and determine bindingcapability, flanked by four framework regions (FRs) which are relativelyconserved in a given species and which putatively provide a scaffoldingfor the CDRs. When nonhuman antibodies are prepared with respect to aparticular antigen, the variable regions can be “reshaped” or“humanized” by grafting CDRs derived from nonhuman antibody on the FRspresent in the human antibody to be modified. Application of thisapproach to various antibodies has been reported by Sato, K. et al.(1993) Cancer Res 53:851-856. Riechmann, L. et al. (1988) “ReshapingHuman Antibodies for Therapy,” Nature 332:323-327; Verhoeyen, M. et al.(1988) “Reshaping Human Antibodies: Grafting An Antilysozyme Activity,”Science 239:1534-1536; Kettleborough, C. A. et al. (1991) “HumanizationOf A Mouse Monoclonal Antibody By CDR-Grafting: The Importance OfFramework Residues On Loop Conformation,” Protein Engineering4:773-3783; Maeda, H. et al. (1991) “Construction Of Reshaped HumanAntibodies With HIV-Neutralizing Activity,” Human Antibodies Hybridoma2:124-134; Gorman, S. D. et al. (1991) “Reshaping A Therapeutic CD4Antibody,” Proc. Natl. Acad. Sci. (U.S.A.) 88:4181-4185; Tempest, P. R.et al. (1991) “Reshaping A Human Monoclonal Antibody To Inhibit HumanRespiratory Syncytial Virus Infection in vivo,” Bio/Technology9:266-271; Co, M. S. et al. (1991) “Humanized Antibodies For AntiviralTherapy,” Proc. Natl. Acad. Sci. (U.S.A.) 88:2869-2873; Carter, P. etal. (1992) “Humanization Of An Anti-p185her2 Antibody For Human CancerTherapy,” Proc. Natl. Acad. Sci. (U.S.A.) 89:4285-4289; and Co, M. S. etal. (1992) “Chimeric And Humanized Antibodies With Specificity For TheCD33 Antigen,” J. Immunol. 148:1149-1154. In some embodiments, humanizedantibodies preserve all CDR sequences (for example, a humanized mouseantibody which contains all six CDRs from the mouse antibodies). Inother embodiments, humanized antibodies have one or more CDRs (one, two,three, four, five, six) which are altered with respect to the originalantibody, which are also termed one or more CDRs “derived from” one ormore CDRs from the original antibody. The ability to humanize an antigenis well known (see, e.g., U.S. Pat. Nos. 5,225,539; 5,530,101;5,585,089; 5,859,205; 6,407,213; 6,881,557).

In one embodiment, the antibody of the invention is a human antibody.Suitable human antibodies may be generated using transgenic ortranschromosomal mice carrying parts of the human immune system ratherthan the mouse system. Such transgenic and transchromosomic mice includemice referred to herein as HuMAb mice and KM mice, respectively, and arecollectively referred to herein as “transgenic mice.”.

The HuMAb mouse contains a human immunoglobulin gene minilocus thatencodes unrearranged human heavy variable and constant (μ and Y) andlight variable and constant (K) chain immunoglobulin sequences, togetherwith targeted mutations that inactivate the endogenous μ and K chainloci (Lonberg, N. et al., Nature 368, 856-859 (1994)). Accordingly, suchmice exhibit reduced expression of mouse IgM or IgK and in response toimmunization, the introduced human heavy and light chain transgenes,undergo class switching and somatic mutation to generate high affinityhuman IgG, κ monoclonal antibodies (Lonberg, N. et al. (1994), supra;reviewed in Lonberg, N., Handbook of Experimental Pharmacology 113,49-101 (1994), Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 1365-93 (1995) and Harding, F. and Lonberg, N., Ann. N. Y. Acad. Sci 764536-546 (1995)). The preparation of HuMAb mice is described in detail inTaylor, L. et al., Nucleic Acids Research 20, 6287-6295 (1992), Chen, J.et al., International Immunology 5, 647-656 (1993), Tuaillon et al., J.Immunol. 152, 2912-2920 (1994), Taylor, L. et al., InternationalImmunology 6, 579-591 (1994), Fishwild, D. et al., Nature Biotechnology14, 845-851 (1996). See also U.S. Pat. Nos. 5,545,806, 5,569,825,5,625,126, 5,633,425, 5,789,650, 5,877,397, 5,661,016, 5,814,318,5,874,299, 5,770,429, 5,545,807, WO 98/24884, WO 94/25585, WO 93/1227,WO 92/22645, WO 92/03918 and WO 01/09187.

The HCo7 mice have a JKD disruption in their endogenous light chain(kappa) genes (as described in Chen et al., EMBO J. 12, 821-830 (1993)),a CMD disruption in their endogenous heavy chain genes (as described inExample 1 of WO 01/14424), a KCo5 human kappa light chain transgene (asdescribed in Fishwild et al., Nature Biotechnology 14:845-851 (1996)),and a HCo7 human heavy chain transgene (as described in U.S. Pat. No.5,770,429).

The HCol2 mice have a JKD disruption in their endogenous light chain(kappa) genes (as described in Chen et al., EMBO J. 12, 821-830 (1993)),a CMD disruption in their endogenous heavy chain genes (as described inExample 1 of WO 01/14424), a KCo5 human kappa light chain transgene (asdescribed in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)),and a HCol2 human heavy chain transgene (as described in Example 2 of WO01/14424).

In the KM mouse strain, the endogenous mouse kappa light chain gene hasbeen homozygously disrupted as described in Chen et al., EMBO J. 12,811-820 (1993) and the endogenous mouse heavy chain gene has beenhomozygously disrupted as described in Example 1 of WO 01/09187. Thismouse strain carries a human kappa light chain transgene, KCo5, asdescribed in Fishwild et al., Nature Biotechnology 14, 845-851 (1996).This mouse strain also carries a human heavy chain transchromosomecomposed of chromosome 14 fragment hCF (SC20) as described in WO02/43478.

Splenocytes from these transgenic mice may be used to generatehybridomas that secrete human monoclonal antibodies according towell-known techniques. Human monoclonal or polyclonal antibodies of thepresent invention, or antibodies of the present invention originatingfrom other species may also be generated transgenically through thegeneration of another non-human mammal or plant that is transgenic forthe immunoglobulin heavy and light chain sequences of interest andproduction of the antibody in a recoverable form therefrom. Inconnection with the transgenic production in mammals, antibodies may beproduced in, and recovered from, the milk of goats, cows, or othermammals. See for instance U.S. Pat. Nos. 5,827,690, 5,756,687, 5,750,172and 5,741,957.

In some antibodies only part of a CDR, namely the subset of CDR residuesrequired for binding, termed the SDRs, are needed to retain binding in ahumanized antibody. CDR residues not contacting antigen and not in theSDRs can be identified based on previous studies (for example residuesH60-H65 in CDR H2 are often not required), from regions of Kabat CDRslying outside Chothia hypervariable loops (see, Kabat et al. (1992)SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, National Institutes ofHealth Publication No. 91-3242; Chothia, C. et al. (1987) “CanonicalStructures For The Hypervariable Regions Of Immunoglobulins,” J. Mol.Biol. 196:901-917), by molecular modeling and/or empirically, or asdescribed in Gonzales, N. R. et al. (2004) “SDR Grafting Of A MurineAntibody Using Multiple Human Germline Templates To Minimize ItsImmunogenicity,” Mol. Immunol. 41:863-872. In such humanized antibodiesat positions in which one or more donor CDR residues is absent or inwhich an entire donor CR is omitted, the amino acid occupying theposition can be an amino acid occupying the corresponding position (byKabat numbering) in the acceptor antibody sequence. The number of suchsubstitutions of acceptor for donor amino acids in the CDRs to includereflects a balance of competing considerations. Such substitutions arepotentially advantageous in decreasing the number of mouse amino acidsin a humanized antibody and consequently decreasing potentialimmunogenicity. However, substitutions can also cause changes ofaffinity, and significant reductions in affinity are preferably avoided.Positions for substitution within CDRs and amino acids to substitute canalso be selected empirically.

The fact that a single amino acid alteration of a CDR residue can resultin loss of functional binding (Rudikoff, S. etc. (1982) “Single AminoAcid Substitution Altering Antigen-Binding Specificity,” Proc. Natl.Acad. Sci. (USA) 79(6):1979-1983) provides a means for systematicallyidentifying alternative functional CDR sequences. In one preferredmethod for obtaining such variant CDRs, a polynucleotide encoding theCDR is mutagenized (for example via random mutagenesis or by asite-directed method (e.g., polymerase chain-mediated amplification withprimers that encode the mutated locus)) to produce a CDR having asubstituted amino acid residue. By comparing the identity of therelevant residue in the original (functional) CDR sequence to theidentity of the substituted (non-functional) variant CDR sequence, theBLOSUM62.iij substitution score for that substitution can be identified.The BLOSUM system provides a matrix of amino acid substitutions createdby analyzing a database of sequences for trusted alignments (Eddy, S. R.(2004) “Where Did The BLOSUM62 Alignment Score Matrix Come From?,”Nature Biotech. 22(8):1035-1036; Henikoff, J. G. (1992) “Amino acidsubstitution matrices from protein blocks,” Proc. Natl. Acad. Sci. (USA)89:10915-10919; Karlin, S. et al. (1990) “Methods For Assessing TheStatistical Significance Of Molecular Sequence Features By Using GeneralScoring Schemes,” Proc. Natl. Acad. Sci. (USA) 87:2264-2268; Altschul,S. F. (1991) “Amino Acid Substitution Matrices From An InformationTheoretic Perspective,” J. Mol. Biol. 219, 555-565. Currently, the mostadvanced BLOSUM database is the BLOSUM62 database (BLOSUM62.iij). Table1 presents the BLOSUM62.iij substitution scores (the higher the scorethe more conservative the substitution and thus the more likely thesubstitution will not affect function). If an antigen-binding fragmentcomprising the resultant CDR fails to bind to ROR1, for example, thenthe BLOSUM62.iij substitution score is deemed to be insufficientlyconservative, and a new candidate substitution is selected and producedhaving a higher substitution score. Thus, for example, if the originalresidue was glutamate (E), and the non-functional substitute residue washistidine (H), then the BLOSUM62.iij substitution score will be 0, andmore conservative changes (such as to aspartate, asparagine, glutamine,or lysine) are preferred.

TABLE 1 A R N D C Q E G H I L K M F P S T W Y V A +4 −1 −2 −2 0 −1 −1 0−2 −1 −1 −1 −1 −2 −1 +1 0 −3 −2 0 R −1 +5 0 −2 −3 +1 0 −2 0 −3 −2 +2 −1−3 −2 −1 −1 −3 −2 −3 N −2 0 +6 +1 −3 0 0 0 +1 −3 −3 0 −2 −3 −2 +1 0 −4−2 −3 D −2 −2 +1 +6 −3 0 +2 −1 −1 −3 −4 −1 −3 −3 −1 0 −1 −4 −3 −3 C 0 −3−3 −3 +9 −3 −4 −3 −3 −1 −1 −3 −1 −2 −3 −1 −1 −2 −2 −1 Q −1 +1 0 0 −3 +5+2 −2 0 −3 −2 +1 0 −3 −1 0 −1 −2 −1 −2 E −1 0 0 +2 −4 +2 +5 −2 0 −3 −3+1 −2 −3 −1 0 −1 −3 −2 −2 G 0 −2 0 −1 −3 −2 −2 +6 −2 −4 −4 −2 −3 −3 −2 0−2 −2 −3 −3 H −2 0 +1 −1 −3 0 0 −2 +8 −3 −3 −1 −2 −1 −2 −1 −2 −2 +2 −3 I−1 −3 −3 −3 −1 −3 −3 −4 −3 +4 +2 −3 +1 0 −3 −2 −1 −3 −1 +3 L −1 −2 −3 −4−1 −2 −3 −4 −3 +2 +4 −2 +2 0 −3 −2 −1 −2 −1 +1 K −1 +2 0 −1 −3 +1 +1 −2−1 −3 −2 +5 −1 −3 −1 0 −1 −3 −2 −2 M −1 −1 −2 −3 −1 0 −2 −3 −2 +1 +2 −1+5 0 −2 −1 −1 −1 −1 +1 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 +6 −4 −2 −2+1 +3 −1 P −1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 +7 −1 −1 −4 −3 −2 S+1 −1 +1 0 −1 0 0 0 −1 −2 −2 0 −1 −2 −1 +4 +1 −3 −2 −2 T 0 −1 0 −1 −1 −1−1 −2 −2 −1 −1 −1 −1 −2 −1 +1 +5 −2 −2 0 W −3 −3 −4 −4 −2 −2 −3 −2 −2 −3−2 −3 −1 +1 −4 −3 −2 +11 +2 −3 Y −2 −2 −2 −3 −2 −1 −2 −3 +2 −1 −1 −2 −1+3 −3 −2 −2 +2 +7 −1 V 0 −3 −3 −3 −1 −2 −2 −3 −3 +3 +1 −2 +1 −1 −2 −2 0−3 −1 +4

The invention thus contemplates the use of guided or random mutagenesisto identify improved CDRs.

In the context of the present invention, conservative substitutions maybe defined by substitutions within the classes of amino acids reflectedin one or more of the following three tables:

Amino Acid Residue Classes for Conservative Substitutions:

TABLE 2 Acidic Residues Asp (D) and Glu (E) Basic Residues Lys (K), Arg(R), and His (H) Hydrophilic Uncharged Residues Ser (S), Thr (T), Asn(N), and Gln (Q) Aliphatic Uncharged Residues Cly (G), Ala (A), Val (V),Leu (L), and Ile (I) Non-polar Uncharged Residues Cys (C), Met (M), andPro (P) Aromatic Residues Phe (F), Tyr (Y), and Trp (W)

Alternative Conservative Amino Acid Residue Substitution Classes:

TABLE 3 1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y W

Alternative Physical and Functional Classifications of Amino AcidResidues:

TABLE 4 Alcohol Group-Containing S and T Residues Aliphatic Residues I,L, V and M Cycloalkenyl Associated Residues F, H, W and Y HydrophobicResidues A, C, F, G, H, I, L, M, R, T, V, W and Y Negatively ChargedResidues D and E Polar Residues C, D, E, H, K, N, Q, R, S and TPositively Charged Residues H, K and R Small Residues A, C, D, G, N, P,S, T and V Very Small Residues A, G and S Residues Involved In A, C, D,E, G, H, K, N, Q, R, S, Turn Formation P and T Flexible Residues Q, T,K, S, G, P, D, E and R

More conservative substitutions groupings include:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

Additional groups of amino acids may also be formulated using theprinciples described in, e.g., Creighton (1984) Proteins: Structure andMolecular Properties (2d Ed. 1993), W. H. Freeman and Company.

Phage display technology can alternatively be used to increase (ordecrease) CDR affinity. This technology, referred to as affinitymaturation, employs mutagenesis or “CDR walking” and re-selection usesthe target antigen or an antigenic fragment thereof to identifyantibodies having CDRs that bind with higher (or lower) affinity to theantigen when compared with the initial or parental antibody (See, e.g.Glaser et al. (1992) J. Immunology 149:3903). Mutagenizing entire codonsrather than single nucleotides results in a semi-randomized repertoireof amino acid mutations. Libraries can be constructed consisting of apool of variant clones each of which differs by a single amino acidalteration in a single CDR and which contain variants representing eachpossible amino acid substitution for each CDR residue. Mutants withincreased (or decreased) binding affinity for the antigen can bescreened by contacting the immobilized mutants with labeled antigen. Anyscreening method known in the art can be used to identify mutantantibodies with increased or decreased affinity to the antigen (e.g.,ELISA) (See Wu et al. 1998, Proc. Natl. Acad. Sci. (U.S.A.) 95:6037;Yelton et al., 1995, J. Immunology 155:1994). CDR walking whichrandomizes the Light Chain may be used possible (see, Schier et al.,1996, J. Mol. Bio. 263:551).

Methods for accomplishing such affinity maturation are described forexample in: Krause, J. C. et al. (2011) “An Insertion Mutation ThatDistorts Antibody Binding Site Architecture Enhances Function Of A HumanAntibody,” MBio. 2(1) pii: e00345-10. doi: 10.1128/mBio.00345-10; Kuan,C. T. et al. (2010) “Affinity Matured Anti-Glycoprotein NMB RecombinantImmunotoxins Targeting Malignant Gliomas And Melanomas,” Int. J. Cancer10.1002/ijc.25645; Hackel, B. J. et al. (2010) “Stability And CDRComposition Biases Enrich Binder Functionality Landscapes,” J. Mol.Biol. 401(1):84-96; Montgomery, D. L. et al. (2009) “Affinity MaturationAnd Characterization Of A Human Monoclonal Antibody Against HIV-1 gp41,”MAbs 1(5):462-474; Gustchina, E. et al. (2009) “Affinity Maturation ByTargeted Diversification Of The CDR-H2 Loop Of A Monoclonal Fab DerivedFrom A Synthetic Naïve Human Antibody Library And Directed Against TheInternal Trimeric Coiled-Coil Of Gp41 Yields A Set Of Fabs With ImprovedHIV-1 Neutralization Potency And Breadth,” Virology 393(1):112-119;Finlay, W. J. et al. (2009) “Affinity Maturation Of A Humanized RatAntibody For Anti-RAGE Therapy: Comprehensive Mutagenesis Reveals A HighLevel Of Mutational Plasticity Both Inside And Outside TheComplementarity-Determining Regions,” J. Mol. Biol. 388(3):541-558;Bostrom, J. et al. (2009) “Improving Antibody Binding Affinity AndSpecificity For Therapeutic Development,” Methods Mol. Biol.525:353-376; Steidl, S. et al. (2008) “In Vitro Affinity Maturation OfHuman GM-CSF Antibodies By Targeted CDR-Diversification,” Mol. Immunol.46(1):135-144; and Barderas, R. et al. (2008) “Affinity Maturation OfAntibodies Assisted By In Silico Modeling,” Proc. Natl. Acad. Sci. (USA)105(26):9029-9034.

The term “transgenic non-human animal” refers to a non-human animalhaving a genome comprising one or more human heavy and/or light chaintransgenes or transchromosomes (either integrated or non-integrated intothe animal's natural genomic DNA) and which is capable of expressingfully human antibodies. For example, a transgenic mouse can have a humanlight chain transgene and either a human heavy chain transgene or humanheavy chain transchromosome, such that the mouse produces human anti-Tauantibody when immunized with Tau antigen and/or cells expressing Tau.The human heavy chain transgene may be integrated into the chromosomalDNA of the mouse, as is the case for transgenic mice, for instance HuMAbmice, such as HCo7 or HCol2 mice, or the human heavy chain transgene maybe maintained extrachromosomally, as is the case for transchromosomal KMmice as described in WO 02/43478. Such transgenic and transchromosomalmice (collectively referred to herein as “transgenic mice”) are capableof producing multiple isotypes of human monoclonal antibodies to a givenantigen (such as IgG, IgA, IgM, IgD and/or IgE) by undergoing V-D-Jrecombination and isotype switching.

The use of the antibodies of the present invention, or theirepitope-binding fragments, as Tau imaging probes has great potential dueto their specificity. Because of the general impermeability of theblood-brain barrier, smaller single-chain variable antibody fragments(scFv molecules) have been found to be preferred as in vivo imagingligands to detect Tau lesions. scFv molecules are formed as a fusionprotein of the variable regions of the heavy (H) and light chain (L)domains of an antibody, connected to one another via a short linkerpeptide of from about 10 to about 25 amino acid residues. The linker isusually rich in glycine for flexibility (e.g., GGGGSGGGGSGGGGS (SEQ IDNO:2) (Fisher, A. et al. (2009)“Efficient Isolation Of SolubleIntracellular Single-Chain Antibodies Using The Twin ArginineTranslocation Machinery,” J. Mol. Biol. 385(1):299-311; Bird, R. E. etal. (1988) “Single-Chain Antigen-Binding Proteins,” Science 242:423-426;Huston, J. S. et al. (1988) “Protein Engineering Of Antibody BindingSites: Recovery Of Specific Activity In An Anti-Digoxin Single-Chain FvAnalogue Produced In Escherichia coli,” Proc. Natl. Acad. Sci. (U.S.A.)85:5879-5883), as well as serine or threonine for solubility, and caneither connect the N-terminus of the Heavy Chain Variable Domain withthe C-terminus of the Light Chain Variable Domain VL, or vice versa(Huang, L. et al. (2013) “Single-Chain Fragment Variable PassiveImmunotherapies For Neurodegenerative Diseases,” Int. J. Mol. Sci.14(9): 19109-19127; Ahmad, Z. A. et al. (2012) “scFv Antibody:Principles And Clinical Application,” Clin. Dev. Immunol. 2012:980250;Huhalov, A. et al. (2004) “Engineered Single Chain Antibody FragmentsFor Radioimmunotherapy,” Q. J. Nucl. Med. Mol. Imaging 48(4):279-288).An example of such a linker is GSTSGSGKPGSGEGSTKG (SEQ ID NO:3)(Whitlow, M. et al. (1993) “An Improved Linker For Single-Chain Fv WithReduced Aggregation And Enhanced Proteolytic Stability,” Protein Eng.6:989-995). A particularly preferred linker for the present inventionhas the amino acid sequence (SEQ ID NO:4): SSGGGGSGGGGGGSSRSS.

In order to facilitate purification and/or recovery, the scFv mayinclude a poly histidine (“His-Tag”) (e.g., (SEQ ID NO:5) HHHHHH). Theimidazole side chains of the histidine residues of the His-Tag canengage in reversible coordinative bonds to certain transition metalions, such as Co²⁺, Zn²⁺ and especially Ni⁺². Thus, when His-tagged scFvmolecules are applied to a matrix containing such metal ions, theyspecifically bind to the matrix, while most untagged proteins do not.The scFv may additionally or alternatively include an “HA-Tag” such as(SEQ ID NO:6) GAYPYDVPDYAS. Human influenza hemagglutinin (HA) is asurface glycoprotein required for the infectivity of the human virus.The HA-tag is derived from the human influenza hemagglutinin (HA)surface glycoprotein, and permits detection of the scFv using ananti-HA-Tag antibody (Millipore).

scFv molecules may be expressed directly or as a fusion protein that islinked to an N-terminal leader peptide that is cleaved in order to yieldthe scFv (see, e.g., Huston, J. S. et al. (1988) “Protein Engineering OfAntibody Binding Sites: Recovery Of Specific Activity In An Anti DigoxinSingle-Chain Fv Analogue Produced In Escherichia coli,” Proc. Natl.Acad. Sci. (U.S.A.) 85:5879-5883). For example, the scFv may be fused tothe modified trp LE leader peptide (MLE)), and cleaved away by acidcleavage of the Asp-Pro peptide bond (Piszkiewicz, D. et al. (1970)“Anomalous Cleavage Of Aspartyl-Proline Peptide Bonds During Amino AcidSequence Determinations,” Biochem. Biophys. Res. Commun.40(5):1173-1178; Fraser, K. J. et al. (1972) “Specific Cleavage BetweenVariable And Constant Domains Of Rabbit Antibody Light Chains By DiluteAcid Hydrolysis,” Biochemistry 11(26):4974-4977; Poulsen, K. et al.(1972) “An Active Derivative Of Rabbit Antibody Light Chain Composed OfThe Constant And The Variable Domains Held Together Only By A NativeDisulfide Bond,” Proc. Natl. Acad. Sci. (U.S.A.) 69(9):2495-2499).

In a further embodiment, an scFv can be linked to another scFv (whichmay be the same or different) in order to form a bivalent molecule. Thiscan be done by producing a single peptide chain with two VH and two VLregions, yielding tandem scFv molecules (Xiong, C.-Y. et al. (2006)“Development Of Tumor Targeting Anti-MUC-1 Multimer: Effects Of di-scFvUnpaired Cysteine Location On PEGylation And Tumor Binding,” ProteinEngineering Design and Selection 19(8):359-367; Kufer, P. et al. (2004)“A Revival Of Bispecific Antibodies,” Trends in Biotechnology22(5):238-244). Alternatively, by forming an scFv whose Heavy ChainVariable Domain is separated from its Light Chain Variable Domain by alinker that is too short to permit such domains to complex with oneanother and form an epitope-binding site, one can force two scFvmolecules to dimerize as a diabody (Hollinger, P. et al. (1993)“Diabodies”: Small Bivalent And Bispecific Antibody Fragments,” Proc.Natl. Acad. Sci. (U.S.A.) 90(14):6444-6448). Diabodies have been shownto have dissociation constants up to 40-fold lower than correspondingscFv molecules, meaning that they have a much higher affinity to theirtarget. Consequently, diabody drugs could be dosed much lower than othertherapeutic antibodies and are capable of highly specific targeting oftumors in vivo (Adams, G. P. et al. (1998) “Prolonged in vivo TumourRetention Of A Human Diabody Targeting The Extracellular Domain Of HumanHER2/neu,” Brit. J. Cancer 77(9):1405-1412). Still shorter linkers (oneor two amino acids) lead to the formation of trimers, so-calledtriabodies or tribodies. Tetrabodies have also been produced. Theyexhibit an even higher affinity to their targets than diabodies (LeGall, F. et al. (1999) “Di-, Tri- And Tetrameric Single Chain FvAntibody Fragments Against Human CD19: Effect Of Valency On CellBinding,” FEBS Letters 453(1):164-168). All of these formats can becomposed from variable scFv molecules so as to form dimers, trimers,etc. having specificity for two or more different epitopes (i.e.,bi-specific diabodies, etc.) (Dincq, S. et al. (2001) “Expression AndPurification Of Monospecific And Bispecific Recombinant AntibodyFragments Derived From Antibodies That Block The CD80/CD86-CD28Costimulatory Pathway,” Protein Express. Purificat. 22(1):11-24).

As discussed below, suitable scFv molecules were isolated from librariesof scFv molecules that had been generated from Tau antibody hybridomasusing a combinatorial phage display technology (see, for example, U.S.Pat. Nos. 5,565,332; 5,580,717; 5,733,743; 6,265,150; and Winter, G. etal. (1994) “Making Antibodies By Phage Display Technology,” Annu. Rev.Immunol. 12.433-455). Numerous phospho-Tau-selective scFv molecules wereidentified and their reactivity toward Tau confirmed byimmunoprecipitation, staining of human and mouse tauopathy tissue aswell as affinity assays. Peripheral injection of these scFv moleculesresulted in a strong in vivo brain signal in transgenic tauopathy micebut not in wild-type or amyloid-β plaque mice. The imaging signal wasshown to correlate very well with co-localization of the probe withintraneuronal Tau aggregates. Both were associated with markers ofendosomes, autophagosomes and lysosomes, suggesting their interaction inthese degradation pathways. Such specific antibody-derived imagingprobes have great potential as diagnostic markers for AD and relatedtauopathies.

Such efforts led to the isolation of preferred anti-phospho-Tau 396,404antibody 6B2G12, which was elicited against a peptide having the aminoacid sequence (SEQ ID NO:7): TDHGAEIVYKSPVVSGDTSPRHL, which correspondsto amino acid residues 386-408 of Tau protein (SEQ ID NO:1), Theunderlined serine residues at positions 11 and 19 of SEQ ID NO:7(corresponding to positions 396 and 404 of Tau (SEQ ID NO:1)) arephosphorylated. The employed immunogen contained this peptide, modifiedto contain an N-terminal cysteine residue that was conjugated to keyholelimpet hemocyanin (KLH).

The Light Chain Variable Domain of antibody 6B2G12 has the amino acidsequence (SEQ ID NO: 8) (CDRs are underlined):ELDVQMTQTP LTLSVTIGQP ASISC KSSQS   LLYSNGKTYL N WLLQRPGQS PKRLIY LVSK  LDS GVPDRFT GSGSGTDFTL KISRVEAEDL GVYYC VQGTH   SPLT FGAGTK LELKThe Heavy Chain Variable Domain of antibody 6B2G12 has the amino acidsequence (SEQ ID NO: 9) (CDRs are underlined):LEVQLQQSGP ELVKPGASVK ISCKTS EYTF   TEYTKH WVKQ SHGKSLEWIG  SINPNNGDTY  YNQKFTD KAT LTVDKSSTTA SMELRSLTFE DSAVYYCAM G   DSAWFAY WGQ GTLVTVS

This antibody was used to form the scFv molecule: scFv235.

The Light Chain Variable Domain of scFv235 has the amino acid sequence(SEQ ID NO: 10) (CDRs are underlinedand the difference from the parental antibody is shown in italics): ELDV

MTQTP LTLSVTIGQP ASISC KSSQS   LLYSNGKTYL N WLLQRPGQS PKRLIY LVSK   LDSGVPDRFT GSGSGTDFTL KISRVEAEDL GVYYC VQGTH   SPLT FGAGTK LELKThe Heavy Chain Variable Domain of scFv235 has the amino acid sequence(SEQ ID NO: 11) (CDRs are underlined): LEVQLQQSGP ELVKPGASVK ISCKTS EYTF  TEYTKH WVKQ SHGKSLEWIG  SINPNNGDTY   YNQKFTD KAT LTVDKSSTTASMELRSLTFE DSAVYYCAM G   DSAWFAY WGQ GTLVTVS

Thus, the Light Chain Variable Domain CDR1 of scFv235 and antibody6B2G12 both have the amino acid sequence (SEQ ID NO:12):KSSQSLLYSNGKTYLN.

The Light Chain Variable Domain CDR2 of scFv235 and antibody 6B2G12 bothhave the amino acid sequence (SEQ ID NO:13): LVSKLDS.

The Light Chain Variable Domain CDR3 of scFv235 and antibody 6B2G12 bothhave the amino acid sequence (SEQ ID NO:14): VQGTHSPLT.

The Heavy Chain Variable Domain CDR1 of scFv235 and antibody 6B2G12 bothhave the amino acid sequence (SEQ ID NO:15): EYTFTEYTKH.

The Heavy Chain Variable Domain CDR2 of scFv235 and antibody 6B2G12 bothhave the amino acid sequence (SEQ ID NO:16): SINPNNGDTYYNQKFTD.

The Heavy Chain Variable Domain CDR3 of scFv235 and antibody 6B2G12 bothhave the amino acid sequence (SEQ ID NO:17): GDSAWFAY.

The complete sequence of scFv235 is (SEQ ID NO: 18):ELDVVMTQTP LTLSVTIGQP ASISCKSSQS LLYSNGKTYLNWLLQRPGQS PKRLIYLVSK LDSGVPDRFT GSGSGTDFTLKISRVEAEDL GVYYCVQGTH SPLTFGAGTK LELKSSGGGGSGGGGGGSSR SSLEVQLQQS GPELVKPGAS VKISCKTSEYTFTEYTKHWV KQSHGKSLEW IGSINPNNGD TYYNQKFTDKATLTVDKSST TASMELRSLT FEDSAVYYCA MGDSAWFAYW GQGTLVTVSAwherein amino acid residues 1-114 are the amino acid residues of theLight Chain Variable Domain of scFv235 (SEQ ID NO:10), amino acidresidues 115-132 are the amino acid residues of the linker (SEQ IDNO:4), amino acid residues 133-249 are the amino acid residues of theHeavy Chain Variable Domain of scFv235 (SEQ ID NO:11).

In a preferred embodiment, scFv235 is prepared as a fusion protein thatincludes an N-terminal leader peptide portion having the amino acidsequence (SEQ ID NO:19): IQEEFKMKKTAIAIAVALAGFATVAQAA, and/or aC-terminal sequence peptide portion. The C-terminal sequence peptideportion may include: an antibody constant domain, such as (SEQ IDNO:20): AKTTPPSVTSGQAGQ (Hussein, A. H. et al. (2007) “Construction andCharacterization of Single-Chain Variable Fragment Antibodies Directedagainst the Bordetella pertussis Surface Adhesins FilamentousHemagglutinin and Pertactin,” Infect. Immun. 75(11):5476-5482), aHis-Tag, such as (SEQ ID NO:5): HHHHHH), and/or an HA-Tag such as (SEQID NO:6): GAYPYDVPDYAS, or any combination or sub-combination thereof,and in any order. A preferred C-terminal peptide portion has the aminoacid sequence (SEQ ID NO:21): AKTTPPSVTSGQAGQHHHHHHGAYPYDVPDYAS, andthus includes (in the N-terminus to C-Terminus direction) SEQ ID NO:20,SEQ ID NO:5, and SEQ ID NO:6.

Thus, in a preferred embodiment, the scFv235 fusion protein willcomprise the amino acid sequence of any of SEQ ID Nos:22-26 (in whichthe N-terminal and/or C-Terminal peptide portions of the ScFv fusion areunderlined):

SEQ ID NO: 22 (a fusion of SEQ ID NOs: 19 and 18):IQEEFKMKKT AJAJAVALAG FATVAQAA EL DVVMTQTPLTLSVTIGQPAS ISCKSSQSLL YSNGKTYLNW LLQRPGQSPKRLIYLVSKLD SGVPDRFTGS GSGTDFTLKI SRVEAEDLGVYYCVQGTHSP LTFGAGTKLE LKSSGGGGSG GGGGGSSRSSLEVQLQQSGP ELVKPGASVK ISCKTSEYTF TEYTKHWVKQSHGKSLEWIG SINPNNGDTY YNQKFTDKAT LTVDKSSTTASMELRSLTFE DSAVYYCAMG DSAWFAYWGQ GTLVTVSASEQ ID NO: 23 (a fusion of SEQ ID NOs: 18 and 20):ELDVVMTQTP LTLSVTIGQP ASISCKSSQS LLYSNGKTYLNWLLQRPGQS PKRLIYLVSK LDSGVPDRFT GSGSGTDFTLKISRVEAEDL GVYYCVQGTH SPLTFGAGTK LELKSSGGGGSGGGGGGSSR SSLEVQLQQS GPELVKPGAS VKISCKTSEYTFTEYTKHWV KQSHGKSLEW IGSINPNNGD TYYNQKFTDKATLTVDKSST TASMELRSLT FEDSAVYYCA MGDSAWFAYW GQGTLVTVS A  AKTTPPSVTS GQAGQ SEQ ID NO: 24 (a fusion of SEQ ID NOs: 18 and 21):ELDVVMTQTP LTLSVTIGQP ASISCKSSQS LLYSNGKTYLNWLLQRPGQS PKRLIYLVSK LDSGVPDRFT GSGSGTDFTLKISRVEAEDL GVYYCVQGTH SPLTFGAGTK LELKSSGGGGSGGGGGGSSR SSLEVQLQQS GPELVKPGAS VKISCKTSEYTFTEYTKHWV KQSHGKSLEW IGSINPNNGD TYYNQKFTDKATLTVDKSST TASMELRSLT FEDSAVYYCA MGDSAWFAYW GQGTLVTVS A  KTTPPSVTSG QAGQHHHHHH GAYPYDVPDY ASSEQ ID NO: 25 (a fusion of SEQ ID NOs: 19, 18 and 20):IQEEFKMKKT AJAJAVALAG FATVAQAA EL DVVMTQTPLTLSVTIGQPAS ISCKSSQSLL YSNGKTYLNW LLQRPGQSPKRLIYLVSKLD SGVPDRFTGS GSGTDFTLKI SRVEAEDLGVYYCVQGTHSP LTFGAGTKLE LKSSGGGGSG GGGGGSSRSSLEVQLQQSGP ELVKPGASVK ISCKTSEYTF TEYTKHWVKQSHGKSLEWIG SINPNNGDTY YNQKFTDKAT LTVDKSSTTASMELRSLTFE DSAVYYCAMG DSAWFAYWGQ GTLVTVSA AK TTPPSVTSGQ   AGQSEQ ID NO: 26 (a fusion of SEQ ID NOs: 19, 18 and 21):IQEEFKMKKT AJAJAVALAG FATVAQAA EL DVVMTQTPLTLSVTIGQPAS ISCKSSQSLL YSNGKTYLNW LLQRPGQSPKRLIYLVSKLD SGVPDRFTGS GSGTDFTLKI SRVEAEDLGVYYCVQGTHSP LTFGAGTKLE LKSSGGGGSG GGGGGSSRSSLEVQLQQSGP ELVKPGASVK ISCKTSEYTF TEYTKHWVKQSHGKSLEWIG SINPNNGDTY YNQKFTDKAT LTVDKSSTTASMELRSLTFE DSAVYYCAMG DSAWFAYWGQ GTLVTVSA AK TTPPSVTSGQ   AGQHHHHHHG  AYPYDVPDYA   S

Although scFv are able to transit across the blood-brain barrier,various ancillary approaches may be used to further promote such transit(Huang, L. et al. (2013) “Single-Chain Fragment Variable PassiveImmunotherapies For Neurodegenerative Diseases,” Int. J. Mol. Sci.14(9):19109-19127).

A limited set of proteins and peptides are transported across theblood-brain barrier via receptor-mediated transcytosis (Hervé, F. et al.(2008) “CNS Delivery Via Adsorptive Transcytosis,” AAPS J.10(3):455-472), the three best-studied ligands being insulin,iron-transferrin and LDL-cholesterol (Bickel, U. et al. (2001) “DeliveryOf Peptides And Proteins Through The Blood-Brain Barrier,” Adv. DrugDeliv. Rev. 46:247-279; Tuma, P. L. et al. (2003) “Transcytosis:Crossing Cellular Barriers,” Physiol. Rev. 83:871-932). Thus, transportof an scFv across the blood-brain barrier can be promoted by fusing thescFv to an antibody, or an epitope-binding fragment thereof, that isimmunospecific for a receptor of such ligands (e.g., the human insulinreceptor (HIR), the transferrin receptor (TfR), low density lipoproteinreceptor-related proteins 1 (LRP1) and 2 (LRP2), non-toxic diphtheriatoxin receptor/Heparin binding epidermal growth factor-like growthfactor, etc). The resulting fusion protein can be transported across theblood-brain barrier through its binding to the receptor (Boado, R. J. etal. (2010) “IgG-Single Chain Fv Fusion Protein Therapeutic ForAlzheimer's Disease: Expression In CHO cells And Pharmacokinetics AndBrain Delivery In The Rhesus Monkey,” Biotechnol. Bioeng. 105:627-635;Jones, A. R. et al. (2007) “Blood-Brain Barrier Transport OfTherapeutics Via Receptor-Mediation,” Pharm. Res. 24(9):1759-1771; Wang,Y. Y. et al. (2009) “Receptor-Mediated Therapeutic Transport Across TheBlood-Brain Barrier,” Immunotherapy 1(6):983-993; Lajoie, J. M. et al.(2015) “Targeting Receptor-Mediated Transport For Delivery Of BiologicsAcross The Blood-Brain Barrier,” Annu. Rev. Pharmacol. Toxicol.55:613-631; Pardridge, W. M. (2102) “Drug Transport Across TheBlood-Brain Barrier,” J. Cereb. Blood Flow Metab. 32(11):1959-1972;Bhaskar, S. et al. (2010) “Multifunctional Nanocarriers For Diagnostics,Drug Delivery And Targeted Treatment Across Blood-Brain Barrier:Perspectives On Tracking And Neuroimaging,” Part. Fibre. Toxicol. 7:3pp. 1-25).

The scFv may be augmented to contain a polycationic peptide thatfacilitates adsorptive-mediated transcytosis. Suitable polycationicpeptides include hexamethylene-diamine, putrescine, spermidine andspermine (Hervé, F. et al. (2008) “CNS Delivery Via AdsorptiveTranscytosis,” AAPS J. 10(3):455-472; Kandimalla, K. K. et al. (2006)“Physiological And Biophysical Factors That Influence Alzheimer'sDisease Amyloid Plaque Targeting Of Native And Putrescine Modified HumanAmyloid Beta40,” J. Pharmacol. Exp. Ther. 318:17-25). The scFv may beaugmented to comprise polycationic groups via treatment that amidatessome or all of its carboxylic groups (i.e., the carboxy-terminal group,or the carboxylic side chains of glutamate or aspartate residue(s) ofthe scFv).

Alternatively, the scFv may be augmented to contain a cell-penetratingpeptide (“CPP”) (Rao, K. S. et al. (2009) “Targeting Anti-HIV Drugs ToThe CNS,” Expert Opin. Drug Deliv. 6(8):771-784; Mathupala, S. P. et al.(2009) “Delivery Of Small-Interfering RNA (siRNA) To The Brain,” ExpertOpin. Ther. Pat. 19(2):137-140; Hervé, F. et al. (2008) “CNS DeliveryVia Adsorptive Transcytosis,” AAPS J. 10(3):455-472). Such peptidesinclude the HIV-1 trans-activating transcriptional activator (TAT)peptide, the Herpes Simplex Virus type-1 transcription factor (HSVVP-22) peptide, antennapedia and penetratin (Wadia, J. S. et al. (2004)“Transducible TAT-HA Fusogenic Peptide Enhances Escape Of TAT-FusionProteins After Lipid Raft Macropinocytosis,” Nat. Med. 10:310-315;Richard, J. P. et al. (2003) “Cell-Penetrating Peptides. A ReevaluationOf The Mechanism Of Cellular Uptake,” J. Biol. Chem. 278:585-590;Temsamani, J. et al. (2004) “The Use Of Cell-Penetrating Peptides ForDrug Delivery,” Drug Discov. Today 9:1012-1019).

III. Uses of the Antibodies and Antibody Fragments of the PresentInvention

The present invention relates to the use of Tau-immunospecific,phospho-Tau-selective, antibodies or Tau-immunospecific,phospho-Tau-selective binding fragments thereof to diagnose and/or treatAlzheimer's disease or tauopathy in a subject patient. With respect tosuch diagnostic utility, such uses may involve detecting, in the subject(i.e., in vivo), the presence of a pathological Tau conformer using the6B2G12 antibody of the present invention, or an epitope-binding fragmentthereof (especially scFv235), that has preferably been detectablylabeled (such molecules being collectively referred to herein as thediagnostic molecules of the present invention). Alternatively, such usesmay involve detecting the presence of a pathological Tau conformer exvivo (e.g., in a biopsy sample, or post-mortem) using the diagnosticmolecules of the present invention.

In one embodiment, such Tau-immunospecific, phospho-Tau-selective,antibodies or Tau-immunospecific, phospho-Tau-selective bindingfragments may be humanized antibodies.

With respect to such therapeutic utility, such uses may involve theadministration of a therapeutically effective amount of the 6B2G12antibody of the present invention, or of an epitope-binding fragmentthereof (especially scFv235) to a patient having one or more symptoms ofAlzheimer's disease or such tauopathy, and thus in need of such therapy,or it may involve the administration of a prophylactically effectiveamounts of the 6B2G12 antibody of the present invention, or of anepitope-binding fragment thereof (especially scFv235) to a patient notexhibiting such symptoms, or exhibiting symptoms of mild dementia orpre-tauopathy that is indicative of incipient Alzheimer's disease ortauopathy, such molecules being collectively referred to herein as thetherapeutic molecules of the present invention.

As described supra, the preferred scFv antibody (scFv235) hasspecificity for P-Ser 396/404, however, scFv molecules havingspecificity for other Tau peptide(s), for example, those described in US2008/0050383, may be used in concert with scFv235.

The term “tauopathy,” as used herein, encompasses any neurodegenerativedisease that involves the pathological aggregation of the microtubuleprotein Tau within the brain. Accordingly, in addition to both familialand sporadic Alzheimer's disease, the tauopathies of the presentinvention include, without limitation, frontotemporal dementia,parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclearpalsy, corticobasal degeneration, Pick's disease, progressivesubcortical gliosis, tangle only dementia, diffuse neurofibrillarytangles with calcification, argyrophilic grain dementia, amyotrophiclateral sclerosis parkinsonism-dementia complex, dementia pugilistica,Down syndrome, Gerstmann-Straussler-Scheinker disease,Hallerworden-Spatz disease, inclusion body myositis, Creutzfeld-Jakobdisease, multiple system atropy, Niemann-Pick disease type C, prionprotein cerebral amyloid angiopathy, subacute sclerosingpanencephalitis, myotonic dystrophy, non-guanamian motor neuron diseasewith neurofibrillary tangles, postencephalitic parkinsonism, acutetraumatic brain injury and chronic traumatic encephalopathy.

IV. Production of the Tau-Binding Molecules of the Present Invention

The Tau-binding molecules of the present invention are preferablyproduced via the recombinant expression of a nucleic acid molecule thatencodes their constituent polypeptide chain(s). The invention thusaccordingly also relates to an expression vector encoding such one ormore polypeptide chains of an antibody of the invention or a fragmentthereof.

An expression vector in the context of the present invention may be anysuitable DNA or RNA vector, including chromosomal, non-chromosomal, andsynthetic nucleic acid vectors (a nucleic acid sequence comprising asuitable set of expression control elements). Examples of such vectorsinclude derivatives of SV40, bacterial plasmids, phage DNA, baculovirus,yeast plasmids, vectors derived from combinations of plasmids and phageDNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, ananti-Tau antibody-encoding nucleic acid is comprised in a naked DNA orRNA vector, including, for example, a linear expression element (asdescribed in, for instance, Sykes, K. F. and Johnston S. A. (1999)“Linear Expression Elements: A Rapid, in vivo, Method To Screen For GeneFunctions,” Nat. Biotechnol. 12:355-359), a compacted nucleic acidvector (as described in for instance U.S. Pat. No. 6,077,835 and/or WO00/70087), a plasmid vector such as pBR322, pUC 19/18, or pUC 118/119, a“midge” minimally-sized nucleic acid vector (as described in, forinstance, Schakowski, F. et al. (2001) “A Novel Minimal-Size Vector(MIDGE) Improves Transgene Expression In Colon Carcinoma Cells AndAvoids Transfection Of Undesired DNA,” Mol. Ther. 3, 793-800 (2001)), oras a precipitated nucleic acid vector construct, such as aCaPO₄-precipitated construct (as described in, for instance, WO00/46147, Benvenisty, N. and Reshef, L. (1986) “Direct Introduction OfGenes Into Rats And Expression Of The Genes,” Proc. Natl. Acad. Sci.(U.S.A.) 83:9551-9555, Wigler, M. et al. (1978) “Biochemical Transfer OfSingle-Copy Eucaryotic Genes Using Total Cellular DNA As Donor,” Cell14, 725 (1978), and Corsaro, C. M. and Pearson, M. L. (1981) “EnhancingThe Efficiency Of DNA-Mediated Gene Transfer In Mammalian Cells,”Somatic Cell Genetics 2:603-616). Such nucleic acid vectors and theusage thereof are well known in the art (see for instance U.S. Pat. Nos.5,589,466 and 5,973,972).

In one embodiment, the vector is suitable for expression of an anti-Tauantibody or a Tau-binding fragment thereof in a bacterial cell. Examplesof such vectors include expression vectors such as BlueScript(Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem 264,5503-5509 (1989), pET vectors (Novagen, Madison, Wis.) and the like).

An expression vector may also or alternatively be a vector suitable forexpression in a yeast system. Any vector suitable for expression in ayeast system may be employed. Suitable vectors include, for example,vectors comprising constitutive or inducible promoters such as alphafactor, alcohol oxidase and PGH (reviewed in: F. Ausubel et al., ed.Current Protocols in Molecular Biology, Greene Publishing and WileyInterScience New York (1987), and Bitter, G. A. et al. (1987)“Expression And Secretion Vectors For Yeast,” Methods Enzymol.153:516-544).

In an expression vector of the invention, an anti-Tau antibody-encodingnucleic acid molecule (or a nucleic acid molecule encoding a Tau-bindingfragment thereof) may comprise or be associated with any suitablepromoter, enhancer, and other expression-facilitating elements. Examplesof such elements include strong expression promoters (e.g., human CMV IEpromoter/enhancer as well as RSV, SV40, SL3-3, MMTV, and HIV LTRpromoters), effective poly (A) termination sequences, an origin ofreplication for plasmid product in E. coli, an antibiotic resistancegene as selectable marker, and/or a convenient cloning site (e.g., apolylinker). Nucleic acids may also comprise an inducible promoter asopposed to a constitutive promoter such as CMV IE (the skilled artisanwill recognize that such terms are actually descriptors of a degree ofgene expression under certain conditions).

In an even further aspect, the invention relates to a recombinanteukaryotic or prokaryotic host cell, such as a transfectoma, whichproduces an antibody of the invention as defined herein or a bispecificmolecule of the invention as defined herein. Examples of host cellsinclude yeast, bacteria, and mammalian cells, such as CHO or HEK cells.For example, in one embodiment, the present invention provides a cellcomprising a nucleic acid stably integrated into the cellular genomethat comprises a sequence coding for expression of an anti-Tau antibodyof the present invention or a Tau-binding fragment thereof. In anotherembodiment, the present invention provides a cell comprising anon-integrated nucleic acid, such as a plasmid, cosmid, phagemid, orlinear expression element, which comprises a sequence coding forexpression of an anti-Tau antibody of the present invention, or suchfragment thereof.

In a further aspect, the invention relates to a method for producing ananti-Tau antibody of the present invention, said method comprising thesteps of a) culturing a hybridoma or a host cell of the invention asdescribed herein above, and b) purifying the antibody of the inventionfrom the culture media.

In general, the produced anti-Tau antibodies and Tau-binding fragmentsthereof may be modified by inclusion of any suitable number of modifiedamino acids and/or associations with such conjugated substituents.Suitability in this context is generally determined by the ability to atleast substantially retain anti-Tau selectivity and/or the anti-Tauspecificity associated with the non-derivatized parent anti-Tauantibody. The inclusion of one or more modified amino acids may beadvantageous in, for example, increasing polypeptide serum half-life,reducing polypeptide antigenicity, or increasing polypeptide storagestability. Amino acid(s) are modified, for example, co-translationallyor post-translationally during recombinant production (e.g., N-linkedglycosylation at N-X-S/T motifs during expression in mammalian cells) ormodified by synthetic means. Non-limiting examples of a modified aminoacid include a glycosylated amino acid, a sulfated amino acid, aprenylated (e. g., farnesylated, geranylgeranylated) amino acid, anacetylated amino acid, an acylated amino acid, a PEGylated amino acid, abiotinylated amino acid, a carboxylated amino acid, a phosphorylatedamino acid, and the like. References adequate to guide one of skill inthe modification of amino acids are replete throughout the literature.Example protocols are found in Walker (1998) Protein Protocols OnCD-Rom, Humana Press, Totowa, N.J. The modified amino acid may, forinstance, be selected from a glycosylated amino acid, a PEGylated aminoacid, a farnesylated amino acid, an acetylated amino acid, abiotinylated amino acid, an amino acid conjugated to a lipid moiety, oran amino acid conjugated to an organic derivatizing agent.

As indicated above, when it is desired to increase the half-life of anadministered therapeutic molecule of the present invention, suchmolecules may be formed to comprise carbohydrate moieties, such aspolyoxyethylated polyols or polyethylene glycol (PEG) (e.g., a PEG witha molecular weight of between about 1,000 and about 40,000, such asbetween about 2,000 and about 20,000, e.g., about 3,000-12,000 g/mol)(Moosmann, A. et al. (2014) “Purification Of PEGylated Proteins, WithThe Example Of PEGylated Lysozyme and PEGylated scFv,” Methods Mol.Biol. 1129:527-538; Jevsevar, S. et al. (2010) “PEGylation OfTherapeutic Proteins,” Biotechnol. J. 5:113-228), or throughglycosylation or by adding or associating proteins such as human serumalbumin (Müller, M. R. et al. (2012) “Improving The PharmacokineticProperties Of Biologics By Fusion To An Anti-HSA Shark VNAR Domain,”MAbs. 4(6):673-685; Stork, R. et al. (2008) “N-Glycosylation As NovelStrategy To Improve Pharmacokinetic Properties Of BispecificSingle-Chain Diabodies,” J. Biol. Chem. 283:7804-7812; Alt, M. et al.(1999) “Novel Tetravalent And Bispecific IgG-like Antibody MoleculesCombining Single-Chain Diabodies With The Immunoglobulin Gamma1 Fc orCH3 Region,” FEBS Lett. 454:90-94; Peters T. et al. (1985) “SerumAlbumin,” Adv. Protein Chem. 37:161-245). Illustrative polymers andmethods to attach them to peptides, are known, (see, for example, U.S.Pat. Nos. 4,766,106; 4,179,337; 4,495,285 and 4,609,546).

V. Pharmaceutical Compositions of the Present Invention

The Tau-binding molecules of the present invention are oftenadministered as pharmaceutical compositions comprising an activetherapeutic agent and a variety of other pharmaceutically acceptablecomponents. See REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (21^(st)Edition) (2005) (Troy, D. B. et al. (Eds.) Lippincott Williams & Wilkins(Publs.), Baltimore Md.), which is hereby incorporated by reference inits entirety. The preferred form depends on the intended mode ofadministration and therapeutic application. The compositions can alsoinclude, depending on the formulation desired, pharmaceuticallyacceptable, non-toxic carriers, excipients, diluents, fillers, salts,buffers, detergents (e.g., a nonionic detergent, such as Tween-20 orTween-80), stabilizers (e.g., sugars or protein-free amino acids),preservatives, tissue fixatives, solubilizers, and/or other materialssuitable for inclusion in a pharmaceutical composition, and which arevehicles commonly used to formulate pharmaceutical compositions foranimal or human administration. The diluent is selected to not to affectthe biological activity of the combination. Examples of such diluentsare distilled water, physiological phosphate-buffered saline, Ringer'ssolutions, dextrose solution, and Hank's solution. In addition, thepharmaceutical composition or formulation may also include othercarriers, or non-toxic, nontherapeutic, non-immunogenic stabilizers andthe like. Examples of suitable aqueous and nonaqueous carriers which maybe employed in the pharmaceutical compositions of the present inventioninclude water, saline, phosphate buffered saline, ethanol, dextrose,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), and suitable mixtures thereof, vegetable oils, such as oliveoil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethylcellulose colloidal solutions, tragacanth gum and injectable organicesters, such as ethyl oleate, and/or various buffers. Other carriers arewell known in the pharmaceutical arts.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe present invention is contemplated.

The compositions may also include large, slowly metabolizedmacromolecules, such as proteins, polysaccharides like chitosan,polylactic acids, polyglycolic acids and copolymers (e.g., latexfunctionalized sepharose, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (e.g., oildroplets or liposomes). Suitability for carriers and other components ofpharmaceutical compositions is determined based on the lack ofsignificant negative impact on the desired biological properties of thechosen compound or pharmaceutical composition of the present invention(e.g., less than a substantial impact (10% or less relative inhibition,5% or less relative inhibition, etc.)) on antigen binding.

The pharmaceutical compositions of the present invention may alsocomprise pharmaceutically acceptable antioxidants for instance (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

The pharmaceutical compositions of the present invention may alsocomprise isotonicity agents, such as sugars, polyalcohols, such asmannitol, sorbitol, glycerol or sodium chloride in the compositions.

The pharmaceutical compositions of the present invention may alsocontain one or more adjuvants appropriate for the chosen route ofadministration such as preservatives, wetting agents, emulsifyingagents, dispersing agents, preservatives or buffers, which may enhancethe shelf life or effectiveness of the pharmaceutical composition. Thecompounds of the present invention may be prepared with carriers thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Such carriers may include gelatin,glyceryl monostearate, glyceryl distearate, biodegradable, biocompatiblepolymers such as ethylene vinyl acetate, polyanhydrides, polyglycolicacid, collagen, polyorthoesters, and polylactic acid alone or with awax, or other materials well known in the art. Methods for thepreparation of such formulations are generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In one embodiment, the compounds of the present invention may beformulated to ensure proper distribution in vivo. Pharmaceuticallyacceptable carriers for parenteral administration include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is known in the art. Except insofar as any conventional mediaor agent is incompatible with the active compound, use thereof in thepharmaceutical compositions of the present invention is contemplated.Supplementary active compounds may also be incorporated into thecompositions.

Pharmaceutical compositions for injection must typically be sterile andstable under the conditions of manufacture and storage. The compositionmay be formulated as a solution, microemulsion, liposome, or otherordered structure suitable to high drug concentration. The carrier maybe a aqueous or nonaqueous solvent or dispersion medium containing forinstance water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. The proper fluidity may be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as glycerol, mannitol,sorbitol, or sodium chloride in the composition. Prolonged absorption ofthe injectable compositions may be brought about by including in thecomposition an agent that delays absorption, for example, monostearatesalts and gelatin. Sterile injectable solutions may be prepared byincorporating the active compound in the required amount in anappropriate solvent with one or a combination of ingredients e.g. asenumerated above, as required, followed by sterilizationmicrofiltration. Generally, dispersions are prepared by incorporatingthe active compound into a sterile vehicle that contains a basicdispersion medium and the required other ingredients e.g. from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Sterile injectable solutions may be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

For parenteral administration, agents of the present invention aretypically formulated as injectable dosages of a solution or suspensionof the substance in a physiologically acceptable diluent with apharmaceutical carrier that can be a sterile liquid such as water, oil,saline, glycerol, or ethanol. Additionally, auxiliary substances, suchas wetting or emulsifying agents, surfactants, pH buffering substancesand the like can be present in compositions. Other components ofpharmaceutical compositions are those of petroleum, animal, vegetable,or synthetic origin. Peanut oil, soybean oil, and mineral oil are allexamples of useful materials. In general, glycols, such as propyleneglycol or polyethylene glycol, are preferred liquid carriers,particularly for injectable solutions. Agents of the invention can beadministered in the form of a depot injection or implant preparationwhich can be formulated in such a manner as to permit a sustainedrelease of the active ingredient. An exemplary composition comprisesscFv235 at about 5 mg/mL, formulated in aqueous buffer consisting of 50mM L-histidine, 150 mM NaCl, adjusted to pH 6.0 with HCl.

Typically, compositions are thus prepared as injectables, either asliquid solutions or suspensions; solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection can also beprepared. The preparation also can be emulsified or encapsulated inliposomes or micro particles, such as polylactide, polyglycolide, orcopolymer, for enhanced adjuvant effect (Langer, G. et al. (1990) “NewMethods of Drug Delivery,” Science 249:1527-1533; Langer, G. et al.(1997) “New Advances In Microsphere-Based Single-Dose Vaccines,”Advanced Drug Delivery Reviews 28:97-119 (1997), which are herebyincorporated by reference in their entirety). Additional formulationssuitable for other modes of administration include oral, intranasal, andpulmonary formulations, suppositories, and transdermal applications.

VI. Administration of the Pharmaceutical Compositions of the PresentInvention

The molecules of the present invention can be administered byparenteral, topical, oral or intranasal means for prophylactic and/ortherapeutic treatment. Intramuscular injection (for example, into thearm or leg muscles) and intravenous infusion are preferred methods ofadministration of the molecules of the present invention. In somemethods, such molecules are administered as a sustained releasecomposition or device, such as a Medipad™ device (Elan Pharm.Technologies, Dublin, Ireland). In some methods, the molecules of thepresent invention are injected directly into a particular tissue wheredeposits have accumulated, for example intracranial injection.

In one embodiment, a pharmaceutical composition of the present inventionis administered parenterally. The phrases “parenteral administration”and “administered parenterally” as used herein denote modes ofadministration other than enteral and topical administration, usually byinjection, and include epidermal, intravenous, intramuscular,intraarterial, intrathecal, intracapsular, intracranial, intraorbital,intracardiac, intradermal, intraperitoneal, intratendinous,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, intracranial, intrathoracic, epidural andintrasternal injection, subcutaneous and infusion. In one embodimentthat pharmaceutical composition is administered by intravenous orsubcutaneous injection or infusion.

In prophylactic applications, pharmaceutical compositions or medicamentsare administered to a patient susceptible to, or otherwise at risk of,Alzheimer's disease in an amount sufficient to eliminate or reduce therisk, lessen the severity, or delay the outset of the disease, includingbiochemical, histologic and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presented duringdevelopment of the disease.

In therapeutic applications (i.e., in applications involving a patientwho has been diagnosed as having Alzheimer's disease or other tauopathy)the therapeutic molecules of the present invention are administered tosuch patient in an amount sufficient to cure, treat, or at leastpartially arrest, the symptoms of the disease (as adduced bybiochemical, histologic and/or behavioral assessment), including itscomplications and intermediate pathological phenotypes in development ofthe disease. In some embodiments, the administration of the therapeuticmolecules of the present invention reduces or eliminates mild cognitiveimpairment in patients that have not yet developed characteristicAlzheimer's pathology.

Effective doses of the provided therapeutic molecules of the presentinvention, for the treatment of the above-described conditions may varydepending upon many different factors, including means ofadministration, target site, physiological state of the patient, othermedications administered, and whether treatment is prophylactic ortherapeutic. Treatment dosages are typically titrated to optimize theirsafety and efficacy. On any given day that a dosage is given, the dosagemay range from about 0.0001 to about 100 mg/kg, and more usually fromabout 0.01 to about 5 mg/kg, of the host body weight. For example,dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within therange of 1-10 mg/kg body weight. Exemplary dosages thus include: fromabout 0.1 to about 10 mg/kg/body weight, from about 0.1 to about 5mg/kg/body weight, from about 0.1 to about 2 mg/kg/body weight, fromabout 0.1 to about 1 mg/kg/body weight, for instance about 0.15mg/kg/body weight, about 0.2 mg/kg/body weight, about 0.5 mg/kg/bodyweight, about 1 mg/kg/body weight, about 1.5 mg/kg/body weight, about 2mg/kg/body weight, about 5 mg/kg/body weight, or about 10 mg/kg/bodyweight

A physician or veterinarian having ordinary skill in the art may readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the anti-Tau antibody or fragment employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved. In general, a suitabledaily dose of a composition of the present invention will be that amountof the compound which is the lowest dose effective to produce atherapeutic effect. Such an effective dose will generally depend uponthe factors described above. Administration may e.g. be intravenous,intramuscular, intraperitoneal, or subcutaneous, and for instanceadministered proximal to the site of the target. If desired, theeffective daily dose of a pharmaceutical composition may be administeredas two, three, four, five, six or more sub-doses administered separatelyat appropriate intervals throughout the day, optionally, in unit dosageforms. While it is possible for a compound of the present invention tobe administered alone, it is preferable to administer the compound as apharmaceutical composition as described above.

An exemplary treatment regime entails administration once per every twoweeks or once a month or once every 3 to 6 months. In some methods, one,two or more antibodies (or epitope-binding fragments thereof) will beadministered in conjunction with the administration of the therapeuticmolecules of the present invention, in which case the dosage of eachsuch administered molecule falls within the ranges indicated.

The dosage and frequency of administration can vary depending on whetherthe treatment is prophylactic or therapeutic. In prophylacticapplications, a relatively low dosage is administered at relativelyinfrequent intervals over a long period of time. Some patients continueto receive treatment for the rest of their lives. In therapeuticapplications, a relatively high dosage at relatively short intervals issometimes required until progression of the disease is reduced orterminated, and preferably until the patient shows partial or completeamelioration of symptoms of disease. Thereafter, the patent can beadministered such therapeutic molecule using a prophylactic dosageregime.

For therapeutic purposes, the molecules of the present invention areusually administered on multiple occasions. Intervals between singledosages (e.g., a bolus or infusion) can be weekly, monthly, or yearly.In some methods, dosage is adjusted to achieve a plasma concentration of1-1000 μg/ml and in some methods 25-300 μg/ml. Alternatively, thetherapeutic molecules of the present invention can be administered as asustained release formulation, in which case less frequentadministration is required. Dosage and frequency vary depending on thehalf-life of the antibody in the patient. In general, human antibodiesshow the longest half-life, followed by humanized antibodies, chimericantibodies, and nonhuman antibodies. scFv molecules generally have shortserum half-lives.

Another aspect of the present invention is a combination therapy whereinan additional antibody, or an epitope-binding fragment thereof,recognizing the Tau protein, or an immunogenic epitope thereof, isadministered in combination with a therapeutic molecule of the presentinvention. In the case of amyloidogenic diseases such as, Alzheimer'sdisease and Down's syndrome, immune modulation to clear amyloid-beta(Aβ) deposits is an emerging therapy. Immunotherapies targeting Aβ haveconsistently resulted in cognitive improvements. It is likely that Tauand Aβ pathologies are synergistic. Therefore, a combination therapytargeting the clearance of both pathologies at the same time may be moreeffective than targeting each individually. In the case of Parkinson'sDisease and related neurodegenerative diseases, immune modulation toclear aggregated forms of the α-synuclein protein is also an emergingtherapy. A combination therapy which targets the clearance of both Tauand α-synuclein proteins simultaneously may be more effective thantargeting each individually.

VII. Utility of the Tau-Binding Molecules of the Present Invention

A. Diagnostic Utility

Detecting the presence of a pathological Tau conformer in a subjectusing a diagnostic molecule of the present invention can be achieved byobtaining a biological sample from the subject (e.g., blood, urine,cerebral spinal fluid), contacting the biological sample with saiddiagnostic antibody, and detecting binding of the diagnostic molecule toa pathological Tau protein conformer in the sample from the subject.Assays for carrying out the detection of a pathological Tau protein in abiological sample that may be readily adapted to the detection of thediagnostic molecules of the present invention are well known in the artand include, without limitation, ELISA, immunohistochemistry, Westernblot.

Alternatively, detecting the presence of a pathological Tau proteinconformer in a subject using a diagnostic molecule of the presentinvention can be achieved using in vivo imaging techniques. In vivoimaging involves administering to the subject the diagnostic antibodyhaving antigenic specificity for a pathological Tau peptide anddetecting binding of the diagnostic antibody reagent to the pathologicalTau protein conformer in vivo.

The diagnostic molecules of the present invention can be administered byinjection (e.g., intravenous injection, intracarotid injection, etc.)into the body of the patient, or directly into the brain by intracranialinjection. The dosage of such molecule should be from about 0.0001 mg/kgto about 100 mg/kg, and more usually from about 0.01 mg/kg to about 5mg/kg, of the host body weight. For example dosages can be about 1 mg/kgbody weight or about 10 mg/kg body weight or within the range of about1-10 mg/kg.

Typically, the diagnostic molecules of the present invention is labeled,although in some methods, the molecule may be unlabeled and a secondarylabeling agent is used to bind to such molecule (coupled or conjugatedeither directly to the molecule or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art). The choice of label depends on the means of detection. Forexample, a fluorescent label (such as a rare earth chelate (e.g., aeuropium chelate)), a fluorescein-type label (e.g., fluorescein,fluorescein isothiocyanate, 5-carboxyfluorescein, 6-carboxy fluorescein,dichlorotriazinylamine fluorescein), a rhodamine-type label (e.g., ALEXAFLUOR® 568 (Invitrogen), TAMRA® or dansyl chloride), VIVOTAG 680 XLFLUOROCHROME™ (Perkin Elmer), phycoerythrin; umbelliferone, Lissamine; acyanine; a phycoerythrin, Texas Red, BODIPY FL-SE® (Invitrogen) or ananalogue thereof, is suitable for optical detection. Chemoluminescentlabels may be employed (e.g., luminol, luciferase, luciferin, andaequorin). Such diagnosis and detection can also be accomplished bycoupling the diagnostic molecule of the present invention to detectablesubstances including, but not limited to, various enzymes, enzymesincluding, but not limited to, horseradish peroxidase, alkalinephosphatase, beta-galactosidase, or acetylcholinesterase, or toprosthetic group complexes such as, but not limited to,streptavidin/biotin and avidin/biotin. Paramagnetic labels andradioisotopic labels can also be employed, and are preferably detectedusing Positron Emission Tomography (PET) or Single-Photon EmissionComputed Tomography (SPECT). Radiolabels include, but are not limitedto, bismuth (²¹³Bi), carbon (¹¹C, ¹³C, ¹⁴C), chromium (⁵¹Cr), cobalt(⁵⁷Co, ⁶⁰Co), copper (⁶⁴Cu), dysprosium (¹⁶⁵Dy), erbium (¹⁶⁹Er),fluorine (¹⁸F), gadolinium (¹⁵³Gd, ¹⁵⁹Gd), gallium (⁶⁸Ga, ⁶⁷Ga),germanium (⁶⁸Ge), gold (¹⁹⁸Au), holmium (¹⁶⁶Ho) hydrogen (³H), indium(¹¹¹In, ¹¹²In, ¹¹³In, ¹¹⁵In), iodine (¹²¹I, ¹²³I, ¹²⁵I, ¹³¹I), iridium(¹⁹²Ir), iron (⁵⁹Fe), krypton (^(81m)Kr), lanthanium (¹⁴⁰La), lutelium(¹⁷⁷Lu), manganese (⁵⁴Mn), molybdenum (⁹⁹Mo), nitrogen (¹³N, ¹⁵N),oxygen (¹⁵O), palladium (¹⁰³Pd), phosphorus (³²P), potassium (⁴²K),praseodymium (¹⁴²Pr), promethium (¹⁴⁹Pm), rhenium (¹⁸⁶Re, ¹⁸⁸Re),rhodium (¹⁰⁵Rh), rubidium (⁸¹Rb, ⁸²Rb), ruthenium (⁸²Ru, ⁹⁷Ru), samarium(¹⁵³Sm), scandium (⁴⁷Sc), selenium (⁷⁵Se), sodium (²⁴Na), strontium(⁸⁵Sr, ⁸⁹Sr, ⁹²Sr), sulfur (³⁵S), technetium (⁹⁹Tc), thallium (²⁰¹Tl),tin (¹¹³Sn, ¹¹⁷Sn), xenon (¹³³Xe), ytterbium (¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Yb),yttrium (⁹⁰Y) and zinc (⁶⁵Zn); positron emitting metals using variouspositron emission tomographies, and non-radioactive paramagnetic metalions (such as paramagnetic ions of Aluminum (Al), Barium (Ba), Calcium(Ca), Cerium (Ce), Dysprosium (Dy), Erbium (Er), Europium (Eu),Gandolinium (Gd), Holmium (Ho), Iridium (Ir), Lithium (Li), Magnesium(Mg), Manganese (Mn), Molybdenum (M), Neodymium (Nd), Osmium (Os),Oxygen (O), Palladium (Pd), Platinum (Pt), Rhodium (Rh), Ruthenium (Ru),Samarium (Sm), Sodium (Na), Strontium (Sr), Terbium (Tb), Thulium (Tm),Tin (Sn), Titanium (Ti), Tungsten (W), and Zirconium (Zi), andparticularly, Co⁺², CR⁺², Cr⁺³, Cu⁺², Fe⁺², Fe⁺³, Ga⁺³, Mn⁺³, Ni⁺²,Ti⁺³, V⁺³, and V⁺⁴). Methods for preparing radiolabeled amino acids andrelated peptide derivatives are known in the art (see for instanceJunghans et al., in Cancer Chemotherapy and Biotherapy 655-686 (2ndedition, Chafner and Longo, eds., Lippincott Raven (1996)) and U.S. Pat.Nos. 4,681,581; 4,735,210; 5,101,827; 5,102,990; RE 35,500; 5,648,471and 5,697,902. For example, a radioisotope may be conjugated by achloramine T method (Lindegren, S. et al. (1998) “Chloramine-T InHigh-Specific-Activity Radioiodination Of Antibodies UsingN-Succinimidyl-3-(Trimethylstannyl)Benzoate As An Intermediate,” Nucl.Med. Biol. 25(7):659-665; Kurth, M. et al. (1993) “Site-SpecificConjugation Of A Radioiodinated Phenethylamine Derivative To AMonoclonal Antibody Results In Increased Radioactivity Localization InTumor,” J. Med. Chem. 36(9):1255-1261; Rea, D. W. et al. (1990)“Site-Specifically Radioiodinated Antibody For Targeting Tumors,” CancerRes. 50(3 Suppl): 857s-861s).

Diagnosis is performed by comparing the number, size, and/or intensityof labeled pathological Tau conformers, Tau aggregates, and/orneurofibrillary tangles in a sample from the subject or in the subject,to corresponding baseline values. The base line values can represent themean levels in a population of non-diseased individuals. Baseline valuescan also represent previous levels determined in the same subject.

The diagnostic methods described above can also be used to monitor asubject's response to therapy. In this embodiment, detecting thepresence of pathological Tau in a subject is determined prior to thecommencement of treatment. The level of pathological Tau in the subjectat this time point is used as a baseline value. At various times duringthe course of treatment the detection of pathological Tau proteinconformers, Tau aggregates, and/or neurofibrillary tangles is repeated,and the measured values thereafter compared with the baseline values. Adecrease in values relative to baseline signals a positive response totreatment. Values can also increase temporarily in biological fluids aspathological Tau is being cleared from the brain.

The present invention is further directed to a kit for performing theabove-described diagnostic and monitoring methods. Typically, such kitscontain the diagnostic antibody of the present invention. The kit canalso include a detectable label. The diagnostic antibody itself maycontain the detectable label (e.g., fluorescent molecule, biotin, etc.)which is directly detectable or detectable via a secondary reaction(e.g., reaction with streptavidin). Alternatively, a second reagentcontaining the detectable label may be utilized, where the secondreagent has binding specificity for the primary antibody. In adiagnostic kit suitable for measuring pathological Tau protein in abiological sample, the antibodies of the kit may be supplied pre-boundto a solid phase, such as to the wells of a microtiter dish.

B. Therapeutic Utility

The presence of labeled anti-Tau antibodies or their Tau-bindingfragments may be detected in vivo for diagnosis purposes. In oneembodiment, such diagnosis comprises: a) administering to a subject aneffective amount of such labeled molecule; b) waiting for a timeinterval following administration in order to allow the labeled moleculeto concentrate at sites (if any) of aggregated Tau and to allow unboundlabeled molecule to be cleared to a background level; c) determining abackground level; and d) detecting such labeled molecule in the subject,such that detection of labeled molecule above the background level isindicative that the subject has a tauopathy, or is indicative of theseverity of such tauopathy. In accordance with such embodiment, theantibody is labeled with an imaging moiety suitable for detection usinga particular imaging system known to those skilled in the art.Background levels may be determined by various methods known in the art,including comparing the amount of labeled molecule detected to astandard value previously determined for a particular imaging system.Methods and systems that may be used in the diagnostic methods of theinvention include, but are not limited to, computed tomography (CT),whole body scan such as positron emission tomography (PET), magneticresonance imaging (MM), and sonography.

The term “treatment” or “treating” as used herein means ameliorating,slowing or reversing the progress or severity of a disease or disorder,or ameliorating, slowing or reversing one or more symptoms or sideeffects of such disease or disorder. For purposes of this invention,“treatment” or “treating” further means an approach for obtainingbeneficial or desired clinical results, where “beneficial or desiredclinical results” include, without limitation, alleviation of a symptom,diminishment of the extent of a disorder or disease, stabilized (i.e.,not worsening) disease or disorder state, delay or slowing of theprogression a disease or disorder state, amelioration or palliation of adisease or disorder state, and remission of a disease or disorder,whether partial or total, detectable or undetectable.

An “effective amount,” when applied to an antibody of the invention,refers to an amount sufficient, at dosages and for periods of timenecessary, to achieve an intended biological effect or a desiredtherapeutic result including, without limitation, clinical results. Thephrase “therapeutically effective amount” when applied to an antibody ofthe invention is intended to denote an amount of the antibody that issufficient to ameliorate, palliate, stabilize, reverse, slow or delaythe progression of a disorder or disease state, or of a symptom of thedisorder or disease. In an embodiment, the method of the presentinvention provides for administration of the antibody in combinationswith other compounds. In such instances, the “effective amount” is theamount of the combination sufficient to cause the intended biologicaleffect.

As indicated above, one aspect of the present invention relates to amethod of preventing or treating Alzheimer's disease or other tauopathyin a subject via the administration of an effective amount of antibody6B2G12 or of an epitope-binding fragment thereof (especially scFv235) toprevent or treat such Alzheimer's disease or other tauopathy. Suchadministration may be provided in order to promote the clearance of Tauaggregates from the brain of a subject or may be provided in order toslow a tangle-related behavioral phenotype in a subject. Additionally,such administration may be provided prophylactically in order to delay,impede, attenuate or prevent the onset of Alzheimer's disease, or othertauopathy associated with the neurofibrillary tangle. An amount adequateto accomplish therapeutic or prophylactic treatment is defined,respectively, as a therapeutically effective dose or a prophylacticallyeffective dose. In both prophylactic and therapeutic regimes, agents areusually administered in several dosages until a sufficient immuneresponse has been achieved. Typically, the immune response is monitoredand repeated dosages are given if the immune response starts to wane. Atherapeutically effective or prophylactically effective dose of such anantibody or epitope-binding fragment thereof may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the molecule to elicit a desired responsein the subject. A therapeutically effective amount is also one in whichany toxic or detrimental effects of the antibody or antibody portion areoutweighed by the therapeutically beneficial effect.

Patients amenable to treatment include individuals having Alzheimer'sdisease or such other tauopathy who show clinically recognized symptomsor indications of such conditions, as well as patients not presentlyshowing symptoms of such conditions. Although Alzheimer's disease isdefinitively diagnosed only post-mortem biopsy, individuals sufferingfrom Alzheimer's disease are clinically diagnosed using the “Alzheimer'sDisease and Related Disorders Association (“ADRDA”) Criteria (Carrillo,M. C. et al. (2013) “Revisiting The Framework Of The National InstituteOn Aging-Alzheimer's Association Diagnostic Criteria,” AlzheimersDement. 9(5):594-601; Budson, A. E. et al. (2012) “New Criteria ForAlzheimer Disease And Mild Cognitive Impairment: Implications For ThePracticing Clinician,” Neurologist 18(6):356-363; Sarazin, M. et al.(2012) “Clinical And Research Diagnostic Criteria For Alzheimer'sDisease,” Neuroimaging Clin. N. Amer. 22(1):23-32; Husain, M. M. (2005)“Clinical Diagnosis And Management Of Alzheimer's Disease,” NeuroimagingClin. N. Amer. 15(4):767-777; Small, G. W. et al. (1997) “Diagnosis AndTreatment Of Alzheimer Disease And Related Disorders. ConsensusStatement Of The American Association For Geriatric Psychiatry, TheAlzheimer's Association, And The American Geriatrics Society,” JAMA278(16):1363-1371). Such individuals can alternatively be distinguishedfrom those having diseases or conditions that are un-related toAlzheimer's disease or other tauopathy by the presence of correlatedrisk factors (i.e., one or more factors that have been found to possessgreater than 50% coincidence with Alzheimer's disease or such othertauopathy). Such correlated risk factors include the finding that apatient has had relatives who have experienced Alzheimer's disease orsuch other tauopathy, or present a family history ofhypercholesterolemia or atherosclerosis. Such correlated risk factorsparticularly include the finding that a patient possesses one or moregenetic or biochemical markers that have been correlated with (i.e.,found to possess greater than 50% coincidence with) the occurrence ofsuch actual disease. Examples of such genetic markers of risk towardAlzheimer's disease include correlated mutations in the APP gene, forexample, mutations at position 717 and positions 670 and 671 of the APPgene (referred to as the Hardy and Swedish mutations respectively).Other suitable markers of known genetic risk include correlatedmutations in the presenilin genes (PS1 and PS2) and in the ApoE4 gene(Bekris, L. M. et al. (2010) “Genetics of Alzheimer Disease,” J.Geriatr. Psychiatry Neurol. 23(4):213-227).

Such PS1 mutations include the substitutions: R35Q; A79V; V82L; L85P;V89L; V94M; V96F; V97L; F105I; F105L; F105V; L113P; L113Q; Y115C; Y115D;Y115H; T116I; T116N; P117A; P117L; P117R; P117S; E120D; E120D; E120G;E120K; E123K; N135D; N135S; A136G; M139I; M139I; M139K; M139T; M139V;I143F; I143M; I143N; I143T; I143V; M146I; M146I; M146I; M146L; M146L;M146V; T147I; L153V; Y154C; Y154N; H163R; H163Y; W165C; W165G; L166H;L166P; L166R; S169L; S169P; S170F; L171P; L173F; L173W; L174M; L174R;F1755; F177L; F1775; S178P; G183V; E184D; V191A; G206A; G206D; G206S;G206V; G209E; G209R; G209V; 5212Y; I213F; I213L; I213T; H214D; H214Y;G217D; G217R; L219F; L219P; Q222H; Q222R; Q223R; L226F; L226R; I229F;A231T; A231V; M233I; M233L; M233L; M233T; M233V; L235P; L235V; F237I;F237L; K239N; T245P; A246E; L248R; L250S; L250V; Y2565; A260V; V261F;V261L; L262F; C263F; C263R; P264L; G266S; P267L; P267S; R269G; R269H;L271V; V272A; E273A; T274R; R278I; R278K; R278S; R278T; E280A; E280G;L282F; L282R; L282V; P284L; P284S; A285V; L286P; L286V; T291P; E318G;R358Q; 5365A; R377M; G378E; G378V; L381V; G384A; F386S; S390I; V391F;L392P; L392V; G394V; N4055; A409T; C410Y; V412I; L418F; L420R; L424F;L424H; L424R; L424V; A426P; A431E; A431V; A434C; L435F; P436Q; P436S;and I439S.

Such PS2 mutations include the substitutions: R29H; G34S; R62C; R62H;R71W; A85V; T122P; T122R; 5130L; V139M; N141I; L143H; V1481; R163H;M174V; S175C; Y231C; Q228L; M239V; M230I; A252T; P334R; T430M; andD439A.

Such ApoE4 alleles include the ε4 allele, ε3 allele and ε2 allele(Verghese, P. B. et al. (2011) “Apolipoprotein E In Alzheimer's DiseaseAnd Other Neurological Disorders,” Lancet Neurol. 10(3):241-252).

In addition, a number of diagnostic tests are available for identifyingindividuals who have Alzheimer's disease. These include measurement ofCSF Tau and Aβ42 levels. Elevated Tau and decreased Aβ42 levels signifythe presence of Alzheimer's disease.

In the case of Alzheimer's disease, virtually anyone is at risk ofsuffering from Alzheimer's disease. Therefore, the therapeutic moleculesof the present invention can be administered prophylactically to thegeneral population without the need for any assessment of the risk ofthe subject patient. The present methods are especially useful for theprophylactic treatment of individuals who do have a known genetic riskof Alzheimer's disease. In asymptomatic patients, treatment can begin atany age (e.g., 10, 20, 30). Usually, however, it is not necessary tobegin treatment until a patient reaches 40, 50, 60, 70, 80 or 90 yearsof age. Treatment typically entails the administration of multipledosages over a period of time. Treatment can be monitored by assayingantibody, or activated T-cell or B-cell responses to the therapeuticagent over time. If the response falls, a booster dosage is indicated.In the case of potential Down's syndrome patients, treatment can beginante-natally by administering the therapeutic agent to the mother duringpregnancy or shortly after the patient's birth.

The present invention provides:

-   1. A binding molecule that is capable of immunospecifically binding    to phosphorylated Tau with more than 2000 fold greater selectivity    than for non-phosphorylated Tau, wherein the molecule is an antibody    or an epitope-binding fragment thereof.-   2. The embodiment of such binding molecule, wherein the molecule is    an epitope-binding fragment of an antibody.-   3. The embodiment of such epitope-binding fragment of an antibody,    wherein the epitope-binding fragment of an antibody is an scFv or a    diabody.-   4. The embodiment of any of the above-described binding molecules,    wherein the molecule immunospecifically binds to the Tau 396/404    peptide (SEQ ID NO:7): TDHGAEIVYKSPVVSGDTSPRHL, wherein the serine    residues at positions 11 and 19 thereof are phosphorylated.-   5. The embodiment of any of the above-described binding molecules,    wherein the epitope-binding fragment comprises one or more of:    -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ ID        NO:12;    -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ ID        NO:13;    -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ ID        NO:14;    -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID        NO:15;    -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID        NO:16; or    -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID        NO:17.-   6. The embodiment of any of the above-described binding molecules,    wherein the epitope-binding fragment comprises:    -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ ID        NO:12;    -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ ID        NO:13;    -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ ID        NO:14;    -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID        NO:15;    -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID        NO:16; and    -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID        NO:17.-   7. The embodiment of the above-described binding molecule having    -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ ID        NO:12;    -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ ID        NO:13;    -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ ID        NO:14;    -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID        NO:15;    -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID        NO:16; and    -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID        NO:17, wherein the molecule is scFv235 (SEQ ID NO:18).-   8. The embodiment of any of the above-described binding molecules,    which is detectably labeled.-   9. The embodiment of any of the above-described detectably labeled    binding molecules, wherein the detectable label is a fluorescent    label, a chemoluminescent label, a paramagnetic label, a    radioisotopic label or an enzyme label.-   10. (A) The embodiment of any of the above-described detectably    labeled binding molecules for use in the manufacture of a medicament    for detecting or measuring the presence or amount of the    phosphorylated Tau protein in the brain of a recipient subject, or    -   (B) the use of any of the above-described detectably embodiments        of labeled binding molecules for detecting or measuring the        presence or amount of the phosphorylated Tau protein in the        brain of a recipient subject.-   11. (A) The embodiment of any of the above-described detectably    labeled binding molecules for use in the manufacture of a medicament    for detecting or measuring the presence or amount of the    phosphorylated Tau protein in the brain of a recipient subject, or    -   (B) the use of any of the above-described embodiments of        detectably labeled binding molecules for detecting or measuring        the presence or amount of the phosphorylated Tau protein in the        brain of a recipient subject;    -   wherein the detection or measurement comprises in vivo imaging        of the binding molecule bound to the phosphorylated Tau protein.-   12. (A) The embodiment of any of the above-described detectably    labeled binding molecules for use in the manufacture of a medicament    for detecting or measuring the presence or amount of the    phosphorylated Tau protein in the brain of a recipient subject, or    -   (B) the use of any of the above-described embodiments of        detectably labeled binding molecules for detecting or measuring        the presence or amount of the phosphorylated Tau protein in the        brain of a recipient subject;    -   wherein the detection or measurement comprises ex vivo imaging        of the binding molecule bound to the phosphorylated Tau protein.-   13. (A) (1) The embodiment of any of the above-described binding    molecules, which is detectably labeled; or    -   (2) the embodiment of any of the above-described detectably        labeled binding molecules, wherein the detectable label is a        fluorescent label, a chemoluminescent label, a paramagnetic        label, a radioisotopic label or an enzyme label;    -   for use in the manufacture of a medicament for diagnosing        Alzheimer's disease or another tauopathy of a subject; or    -   (B) (1) the use of any of the above-described embodiments of        detectably labeled binding molecules for diagnosing Alzheimer's        disease or another tauopathy of a subject; or        -   (2) the use of any of the above-described embodiments of            detectably labeled binding molecules, wherein the detectable            label is a fluorescent label, a chemoluminescent label, a            paramagnetic label, a radioisotopic label or an enzyme            label, for diagnosing Alzheimer's disease or another            tauopathy of a subject.-   14. (A) (1) The embodiment of any of the above-described binding    molecules, which is detectably labeled; or    -   (2) the embodiment of any of the above-described detectably        labeled binding molecules, wherein the detectable label is a        fluorescent label, a chemoluminescent label, a paramagnetic        label, a radioisotopic label or an enzyme label;    -   for use in the manufacture of a medicament for diagnosing        Alzheimer's disease or another tauopathy of a subject; or    -   (B) (1) the use of any of the above-described embodiments of        detectably labeled binding molecules for diagnosing Alzheimer's        disease or another tauopathy of a subject; or        -   (2) the use of any of the above-described embodiments of            detectably labeled binding molecules, wherein the detectable            label is a fluorescent label, a chemoluminescent label, a            paramagnetic label, a radioisotopic label or an enzyme            label, for diagnosing Alzheimer's disease or another            tauopathy of a subject;    -   wherein the medicament is an in vivo medicament that is        administered to the subject.-   15. (A) (1) The embodiment of any of the above-described binding    molecules, which is detectably labeled; or    -   (2) the embodiment of any of the above-described detectably        labeled binding molecules, wherein the detectable label is a        fluorescent label, a chemoluminescent label, a paramagnetic        label, a radioisotopic label or an enzyme label;    -   for use in the manufacture of a medicament for diagnosing        Alzheimer's disease or another tauopathy of a subject; or    -   (B) (1) the use of any of the above-described embodiments of        detectably labeled binding molecules for diagnosing Alzheimer's        disease or another tauopathy of a subject; or        -   (2) the use of any of the above-described embodiments of            detectably labeled binding molecules, wherein the detectable            label is a fluorescent label, a chemoluminescent label, a            paramagnetic label, a radioisotopic label or an enzyme            label, for diagnosing Alzheimer's disease or another            tauopathy of a subject;    -   wherein the medicament is incubated ex vivo with a biopsy sample        of the subject.-   16. The embodiment of any of such uses, wherein the tauopathy is    selected from the group comprising frontotemporal dementia,    parkinsonism linked to chromosome 17 (FTDP-17), progressive    supranuclear palsy, corticobasal degeneration, Pick's disease,    progressive subcortical gliosis, tangle only dementia, diffuse    neurofibrillary tangles with calcification, argyrophilic grain    dementia, amyotrophic lateral sclerosis parkinsonism-dementia    complex, dementia pugilistica, Down syndrome,    Gerstmann-Straussler-Scheinker disease, Hallerworden-Spatz disease,    inclusion body myositis, Creutzfeld-Jakob disease, multiple system    atropy, Niemann-Pick disease type C, prion protein cerebral amyloid    angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy,    non-guanamian motor neuron disease with neurofibrillary tangles,    postencephalitic parkinsonism, acute traumatic brain injury and    chronic traumatic encephalopathy.

EXAMPLES

The following examples illustrate various methods for compositions inthe diagnostic or treatment methods of the invention. The examples areintended to illustrate, but in no way limit, the scope of the invention.

Example 1 Isolation of scFv235

scFv molecules were generated from hybridoma clone 6B2G12 (raisedagainst P-Ser396/404 of the Tau protein) (Congdon, E. E. et al. (2013)“Antibody Uptake into Neurons Occurs Primarily via Clathrin-dependentFcgamma Receptor Endocytosis and Is a Prerequisite for Acute Tau ProteinClearance,” J. Biol. Chem. 288:35452-35465; Gu, J. et al. (2013) “TwoNovel Tau Antibodies Targeting The 396/404 Region Are Primarily Taken UpBy Neurons And Reduce Tau Protein Pathology,” J. Biol. Chem.288(46):33081-33095). Briefly, the hybridoma cell line 6B2G12 was grownat 37° C. with 5% CO₂ in RPMI medium containing streptomycin (50 μg/ml)and Penicillin G (50 U/ml), and its mRNA was isolated and purified asper the protocol of an RNA isolation kit (Promega), and subsequentlystored at −80° C. The first strand cDNA was constructed as per theprotocol of a first strand cDNA synthesis kit (Takara kit (TAK6115A)).

Clones were screened for their ability to express scFv molecules thatwere immunospecific for the P-Ser396, 404 Tau epitope using peptides(Keck Foundation, Yale University) having the sequences of

Tau-serine 396/404 (SEQ ID NO: 27):  RENAKAKTDHGAEIVYKSPVVSGDTSPRHLand Tau-phospho-serine 396/404 (SEQ ID NO: 28):  RENAKAKTDHGAEIVYK SPVVSGDT S PRHLwherein the underlined serine residues at positions 18 and 26 of SEQ IDNO:28 (corresponding to positions 396 and 404 of Tau (SEQ ID NO:1)) arephosphorylated. These peptides were also used for panning, ELISA and inBiacore for binding studies. Thousands of clones were generated, ofwhich binding of randomly selected 90 clones is depicted in FIGS. 1A-1B.Among such selected scFv molecules was scFv235 (see FIG. 1A).

Production of Soluble Antibodies

scFv were produced as described in Barbas, C. F., III et al. (2001)“Phage Display: A Laboratory Manual,” Cold Spring Harbor Press, Coldspring Harbor, N.Y. Briefly, scFv235 was produced in competentnon-suppressor E. coli cells (Top 10 cells, Invitrogen) in super broth(SB) medium (10 g MOPS, 30 g tryptone, 20 g yeast extract per liter)with 50 μg/ml of carbenicillin and 20 ml of 1M MgCl2 per liter. Theculture was induced by adding 1 mM IPTG (isopropyl-β-D-thiogalactoside)and scFv235 was isolated from the pellet as described in Barbas, C. F.,III et al. (2001).

Example 2 Characterization of scFv235

A single construct of those, scFv235 showed superior phospho-epitopespecificity both when the peptide is coated on ELISA plates (FIG. 2A) aswell as in Biacore when the molecule was immobilized and the bindingpeptides are in solution. Furthermore, scFv235 or its parent antibody,6B2G12, immunoprecipitated Tau protein in brain homogenates ofindividuals who had Alzheimer's disease (FIGS. 2B-2C), and show partialco-localization staining with pathological Tau on fixed Alzheimer's andPick's disease brain tissue (FIG. 2D-2E). Significantly, scFv235detected only full length Tau protein bands, whereas 6B2G12 bound toboth full length and degraded Tau fragments.

The His-tagged scFv235 was purified using Ni-NTA agarose resin loaded ina gravity column, following the Qiagen kit protocol. The antibodyfragment was then dialyzed in PBS and used for further characterization.Phospho-selectivity of the purified scFv was first confirmed in an ELISAassay as described by Asuni, A. A. et al. (2007) “ImmunotherapyTargeting Pathological Tau Conformers In A Tangle Mouse Model ReducesBrain Pathology With Associated Functional Improvements,” J. Neurosci.27:9115-9129, in which Tau-phospho-serine 396/404 and Tau-serine 396/404peptides were coated onto the plate overnight at 4° C., and afterblocking, incubated with 2.5 μg scFv235 for 2 h. HRP conjugated anti-HAsecondary antibody was used to detect bound scFv235 at 450 nm.

Surface Plasmon Resonance (SPR) Analysis

The binding kinetics of scFv235's and its parent antibody, 6B2G12, totheir target molecules were measured by SPR in a Biacore 2000 (GEHealthcare) according to the manufacturer's instructions and asdescribed previously (Krishnaswamy, S. et al. (2009) “Cloning AntifungalSingle Chain Fragment Variable Antibodies By Phage Display AndCompetitive Panning Elution,” Anal. Biochem. 395:16-24; Krishnaswamy, S.et al. (2011) “Isolation And Characterization Of Recombinant SingleChain Fragment Variable Anti-Idiotypic Antibody Specific To Aspergillusfumigatus Membrane Protein,” J. Immunol. Methods 366:60-68). Briefly,scFv235/antibody (10 μg/ml) was diluted in 10 mM sodium acetate, pH 5.0,and was immobilized on a separate CMS sensor chip with an amine couplingkit (7 min contact time at 5 μl/min flow rate). Unreacted surface-boundmaterial was blocked with ethanolamine. One channel of each sensor chip,prepared in the same way without scFv235 or antibody, was used tomonitor the nonspecific binding of the peptide. All measurements weredone with HBS-EP buffer (10 mM HEPES at pH 7.4, 150 mM NaCl, 3.4 mM EDTAand 0.005% surfactant P20) at flow rate of 5 μl/min at 25° C. After eachmeasurement, the chip surface was regenerated with 10 μl of a buffercontaining 500 mM NaCl and 0.1M glycine HCl, pH 8.0. Binding ofTau-phospho-serine 396/404 and Tau-serine 396/404 peptides wasdetermined at various concentrations and the equilibrium dissociationconstant (Kd) was calculated using BIAevaluation software withKd=k_(off)/k_(on). Table 5 and Table 6 summarize the kinetics andspecificity of binding of the 6B2G12 antibody and scFv235.

TABLE 5 K_(D) Affinity (M) Analyte Peptides 6B2G12 scFv235Phospho-Tau-serine 396/404 3.95 × 10⁻¹⁰ 1.04 × 10⁻⁶ Tau-serine 396/4042.51 × 10⁻⁹  4.06 × 10⁻³

The selectivity of scFv235 for the phosphorylated peptide (SEQ ID NO:28)relative to the non-phosphorylated peptide (SEQ ID NO:27)(1/[1.04×10⁻⁶/4.06×10⁻³]) is 3.9×10³.

TABLE 6 Analyte Association (1/Ms) Dissociation (1/S) K_(D) (M) Peptides6B2G12 scFv235 6B2G12 scFv235 6B2G12 scFv235 Phospho-Tau- 3.69 × 10⁶3.93 × 10³ 1.46 × 10⁻³  3.9 × 10⁻³ 3.95 × 10⁻¹⁰ 9.94 × 10⁻⁷ serine396/404 Tau-serine 1.16 × 10⁶ 9.74 2.92 × 10⁻³ 3.96 × 10⁻² 2.51 × 10⁻⁹4.06 × 10⁻³ 396/404

scFv235 thus has an approximately 2,508 fold lower affinity for thephosphorylated P-Ser396/404 peptide than antibody 6B2G12 (i.e., forphospho-Tau-serine 396/404, the ratio of [6B2G12 Association/6B2G12Dissociation] to [scFv235 Association/scFv235 Dissociation]=2,508), andan 1.62 million fold lower affinity for the non-phosphorylatedP-Ser396/404 peptide than antibody 6B2G12 (i.e., for non-phosphorylatedTau 396/404, the ratio of [6B2G12 Association/6B2G12 Dissociation] to[scFv235 Association/scFv235 Dissociation]=1.62 million).

Antibody 6B2G12 thus has a K_(a) for the non-phosphorylated peptide thatis about 3 times lower than its K_(a) for the phosphorylated peptide,and a K_(d) for the non-phosphorylated peptide that is about double itsK_(d) for the phosphorylated peptide. In contrast, and unexpectedly,scFv235, which is derived from this antibody, exhibits a K_(a) for thenon-phosphorylated peptide that is about 400 times lower than its K_(a)for the phosphorylated peptide, and a K_(d) for the non-phosphorylatedpeptide that is about ten times lower than its K_(d) for thephosphorylated peptide.

Immunoprecipitation

Dynabeads His-Tag Isolation and Dynabeads Protein G kits (Invitrogen)were used for immunoprecipitation of Tau in AD brain homogenate (250 μg)with scFv235 or antibody (10 μg), with the protocol as per the kitinstructions. The pulled down proteins were separated on 10% acrylamidegels, followed by Western blotting. The proteins were transferred fromthe gels onto nitrocellulose membranes. The membranes were blocked with5% skimmed milk in PBS for 1 h at room temperature. Then the blot wasincubated with 1:1000 dilution of CP27 (human specific Tau antibody) ortotal Tau antibody (Dako), overnight at 4° C. This was followed by a 2 hincubation with HRP conjugated secondary antibody (1:1000) in PBScontaining 5% skimmed milk. After washing, the proteins on the membranewere detected by enhanced chemiluminescence (ECL; Pierce). Images ofimmunoreactive bands were acquired using the Fuji LAS-4000 imagingsystem.

The scFv molecules specifically pulled down Tau bands in the range of50-70 kDa, bound to pathological Tau in AD and Pick's disease brainsections, and had affinity on Biacore toward the phospho-epitope(1.04×10⁻⁶-6.05×10⁻⁸M) and the non-phospho-epitope (4.06×10⁻³-1.86×10⁻⁸M).

Human Brain Tissue Staining

The His-Tag purified scFv235 was labeled using ALEXA® FLUOR 568 proteinlabeling kit (Molecular Probes, Invitrogen) as per the kit instructions.Several AD, Pick's disease and age-matched control brains (NationalDisease Research Interchange) were stained with the 568-labeled scFv235using standard procedures. Briefly, slide mounted paraffin embeddedbrain sections were deparaffinized in xylene and cleared through anethanol gradient, washed in PBS, followed with epitope unmasking in 0.3%formic acid, and after PBS washes incubated with a mixture of 10 μg/mlof scFv235 and 1:1000 dilution of PHF1 Tau antibody culture supernatantovernight at 4° C. Bound PHF1 was detected with a fluorescently-taggedsecondary antibody (Alexa Fluor 488 goat anti-mouse IgG, 1:500 dilution,Life Technologies). Nuclei were detected with DAPI and the slidescoverslipped with ProLong Gold antifade reagent mounting media (LifeTechnologies). Images were captured on a Fluoview 1000 laser scanningconfocal microscope (Olympus) using wavelength and filters according tothe characteristics of the fluorophores.

Example 3 Animals, Injections of Labeled Antibodies and IVIS Imaging

In light of its superior characteristics, scFv235 was chosen for in vivoimaging in mice. Mice were housed in AAALAC-approved facilities andreceived food and water ad libitum. The experiments were performed underan IACUC-approved protocol. Three different transgenic tangle modelswere used: htau (Andorfer, C. et al. (2003) “Hyperphosphorylation AndAggregation Of Tau In Mice Expressing Normal Human Tau Isoforms,” J.Neurochem. 86:582-590); htau/PS1 (Boutajangout, A. et al. (2010)“Immunotherapy Targeting Pathological Tau Prevents Cognitive Decline InA New Tangle Mouse Model,” J. Neurosci. 30:16559-16566), and JNPL3(Lewis, J. et al. (2000) “Neurofibrillary Tangles, Amyotrophy AndProgressive Motor Disturbance In Mice Expressing Mutant (P301L) TauProtein,” Nat. Genet. 25:402-405). Controls were wild-type mice andtransgenic plaque mice (Tg-SwDI (Davis, J. et al. (2004) “Early-OnsetAnd Robust Cerebral Microvascular Accumulation Of Amyloid Beta ProteinIn Transgenic Mice Expressing Low Levels Of A Vasculotropic Dutch/IowaMutant Form Of Amyloid Beta-Protein Precursor,” J. Biol. Chem.279:20296-20306).

Intracarotid injections were performed as described by Asuni, A. A. etal. (2007) “Immunotherapy Targeting Pathological Tau Conformers In ATangle Mouse Model Reduces Brain Pathology With Associated FunctionalImprovements,” J. Neurosci. 27, 9115-9129. Briefly, mice wereanesthetized with 2% isoflurane and maintained with 1.5% isoflurane in30% 02. After exposing the carotid sheath, left common carotid artery(CCA), external carotid artery (ECA), and internal carotid artery (ICA)were exposed via a midline incision. A silk suture was tied to thedistal end of the ECA, and the left CCA, ICA, and pterygopalatine arterywere temporarily tied. A 30 gauge needle connected to PE-10 tubing(Becton Dickinson, San Diego, Calif.) was attached to a 1 ml syringe and250 μg of labeled scFv235 in 400-500 μl PBS was then administered intothe common carotid artery over a period of 10-15 min. KRAZY® glue wasapplied to the site of injection to prevent postoperative bleeding.Alternatively, mice were injected into the femoral vein using the samedose. The medial side of the right thigh was shaved and cleaned withBetadine solution and 70% ethanol. A small incision (0.5 cm) parallel tothe vein was made on the skin of the internal side of the thigh. Whenthe buttonhole of skin opens, the femoral vein is visible. A 30 gaugeneedle connected to PE-10 tubing was attached to a 1 ml syringe,administered through the needle punctured upward into the femoral veinover a period of 3-5 min manually. Once the needle was removed, the siteof the puncture was compressed with a cotton-tipped applicator, in orderto avoid bleeding. As soon as the blood flow had ceased, the skin wassutured with 4/0 braided silk thread using single interrupted sutures.For IVIS imaging, scFv235 or 6B2G12 was conjugated with VIVOTAG 680 XLFLUOROCHROME™ (Perkin Elmer). The mice were shaved on the head and bodyto avoid light diffraction caused by the fur. After injection, mice weresubjected to imaging, at various intervals as detailed in FIGS. 3A-3D,in IVIS Lumina XR (Perkin Elmer) using an excitation filter of 675 nmand Cy5.5 emission filter.

Intracarotid injection with scFv235 tagged with a fluorophore led to apartial (Tau-5) to complete co-localization (PHF-1) with stainedintraneuronal Tau aggregates within the brain in transgenic tauopathymice (n=6) but not in wt mice (n=4). Furthermore, the scFv co-localizedwith markers of endosomes-autophagosomes-lysosomes, which are known tocontain Tau aggregates, suggesting that this interaction takes place inthese degradation pathways. Preliminary findings from IVIS imaging inlive mice show a substantially stronger signal from the right hemispherein transgenic compared to wild-type mice when imaged about 30 min afterright intracarotid injection. This signal gradually diminishes andspreads from the brain to the periphery over several hours.

Analysis of IVIS Images

Images were analyzed using the Living Imaging software from Perkin Elmeras per online protocol. Total Radiant Efficiency (summed signalintensity) was calculated in the outlined region of interest (brain).Total Radiant Efficiency was calculated as:

${Total}\mspace{14mu} {Radiant}\mspace{14mu} {Efficiency}{= \frac{p\text{/}\sec \text{/}{cm}^{2}\text{/}sr}{\mu \; W\text{/}{cm}^{2}}}$

Immunohistochemistry of Mouse Brain Sections

After imaging, the tissue was processed as described by Asuni, A. A. etal. (2007) “Immunotherapy Targeting Pathological Tau Conformers In ATangle Mouse Model Reduces Brain Pathology With Associated FunctionalImprovements,” J. Neurosci. 27, 9115-9129. Briefly, the mice wereperfused transaortically with PBS, the brains removed and fixed in 2%PLP overnight, placed in 2% DMSO in 20% glycerol phosphate bufferovernight, then sectioned to detect 680XL-scFv235 signal and todetermine scFv subcellular location by co-staining with marker of Tauprotein and the endosomal/autophagosomal/lysosomal system. Serialcoronal 40 μm sections of the brain were prepared and immunofluorescencestaining was performed per standard protocol on free floating sections.Briefly, after PBS washes, 0.3% Triton-X-100 permeabilization, and blockin 5% BSA, tissue was incubated with antibodies (1:500-1:1000) overnightat 4° C. [Tau (Tau5, MC1, PHF1), microglia (Iba1), early endosomes(EEA1), late endosomes (rab7), late endosomes/lysosomes (lamp2, P62),and autophagosomes (LC3 and P62). Bound antibodies were detected with anAlexa Fluor 488 goat anti-mouse/rabbit IgG (Invitrogen), and nuclei withDAPI. After coverslipping with ProLong Gold, the tissue was analyzedwith an LSM 700 Zeiss confocal laser scanning microscope. The extent ofstaining with each label and their co-localization was rated using asemi-quantitative scale of 0-4 4 with 1-4 indicating increasing degreeof these parameters (1: minimal; 2: modest; 3: moderate; 4: extensive).Such analysis was performed for: 1) residual signal of the injectedfluorescently-tagged scFv/antibody; 2) degree of brain Tau pathology;3-4) extent of co-localization of injected scFv/antibody signal with 3)Tau antibody staining or 4) endosome/lysosome/autophagosome antibodystaining.

Statistics

Correlation analysis between the different parameters described in FIGS.6A-6F was analyzed by Spearman rank correlation. When scFv235 orantibody brain tissue signal was one parameter, only mouse brainsremoved within few hours after injection were included, as the braintissue signal weakens over time. For correlations between IVIS signaland Tau pathology, all the injected mice were included.

Imaging Analysis

Imaging studies using In vivo Imaging System (“IVIS”) revealed strongbrain signals from transgenic (“tg”) tauopathy mice whennear-infrared-dye-labeled-scFv235 was injected into the right carotidartery (250 μg/450-500 μl). The signal remained high and relativelystable for at least up to 250-300 min (FIGS. 3A-3D). JNPL3 mice (Lewis,J. et al. (2000) “Neurofibrillary Tangles, Amyotrophy And ProgressiveMotor Disturbance In Mice Expressing Mutant (P301L) Tau Protein,” Nat.Genet. 25:402-405), with the P301L Tau mutation, showed stronger signalthan htau/PS1 mice (Boutajangout, A. et al. (2010) “ImmunotherapyTargeting Pathological Tau Prevents Cognitive Decline In A New TangleMouse Model,” J. Neurosci. 30:16559-16566), which have milder overallTau pathology. Brain signal from scFv235 injected P301L mice peaked at35-38 min (1714% above pre-injection baseline signal), and remainedstrong at 82 and 330 minutes post-injection (1675% and 1468%,respectively). In the htau/PS1 mice, the scFv235 brain signal peaked at37 minutes (1443% above pre-injection baseline signal), and remainedstrong at 65 and 176 minutes post-injection (1144% and 1093%,respectively). The htau/PS1 model had less signal than P301L mice, bothin brain and periphery. Older mice within each genotype had strongersignal than younger mice which is as expected since those should havemore advanced Tau pathology. Minimal signal was observed in wild-type(wt) mice injected with the same probe or in tangle mice that receivedonly the fluorescent dye alone without the scFv. The antibody from whichthe scFv was derived, 6B2G12, provided similar results when injected atthe same dose and via the same route, albeit its signal intensity wassubstantially less. Imaging at 20-95 minutes post-injection showed asignal of 600-632% above baseline value with a modest reduction at thedepicted 170 and 291 minutes (497% and 431%, respectively). Older (11-12months) P301L mice injected with scFv235 had peak brain signal [totalradiant efficiency (TRE)] of 2.23×10¹¹ and 2.64×10¹¹ respectively,whereas the younger mice (3 and 8 months) had lower peak signals of1.88×10¹¹ and 1.86×10¹¹, respectively. The same P301L model (7-10months) had strong but lesser brain signal after 6B2G12 injection,ranging from 2.20×10¹1 to 1.16×10¹¹. Similar signal intensity wasobserved in an old scFv235-injected htau/PS1 mouse (22 months; peak at1.40×10¹¹) but limited in a 7 months old htau/PS1 mouse, which wasconfirmed to lack Tau pathology. Wt mice (12-13 months) and one htaumouse (13 months) had low signal at all time points and had no Taupathology. Injection of the fluorescent-tag alone in an old htau/PS1mouse (23 months) and a P301L mouse (7 months) gave a higher brainsignal than in wt mice, injected with scFv235 or 6B2G12, but it wassubstantially less than in any of the tauopathy mice injected with thescFv or antibody. These two dye injected mice were confirmed to haveextensive Tau pathology.

The brains were subsequently removed for further analyses to verify thepresence and subcellular location of the scFv or antibody in the brain(FIGS. 4A-4D). Both were detected primarily in neurons, partiallyco-localized with pathological Tau proteins and exclusively in theendosome-autophagosome-lysosome system. A smaller portion was detectedin microglia. Transgenic mice with limited Tau pathology, because ofyoung age, model differences or intra-model variability, had a weak IVISbrain signal after the diagnostic ligand injection.

The inventors also assessed the feasibility of using the intravenous(i.v.) route, which would be more useful applicable for multipleinjections and for clinical use. Importantly, strong brain signals werealso observed for both ligands when this route of administration wasused, at the same dose, indicating that even with first pass liverclearance sufficient amount of the ligand entered the brain to allow usto clearly detect Tau lesions. Longer imaging sessions were conducted inthe i.v. injected mice, showing a gradual clearance of the signal over8-14 days (FIG. 3C-3D). As for the intracutaneous (i.c.) injection, miceinjected with the scFv showed generally stronger signal than miceinjected with the Tau antibody. In i.v. injected P301L mice, peakscFv235 brain signal was detected at 18 min (1754% above pre-injectionbaseline), with lesser signal at 210 min (18% reduction from peaksignal), that had substantially subsided at 8 days (43% reduction). In6B2G12 antibody injected mice, brain signal was strong at 25 minutes(1211% above pre-injection baseline), peaked at 35 minutes (1445%), withlesser signal at 120 minutes (11% reduction from peak signal) and wasmuch weaker by 8 and 12 days (67% and 70% reduction, respectively).P301L mice injected with control pooled IgG had a modest IVIS signaldespite having very extensive Tau pathology, and the brain signal didnot co-localize with Tau markers. This is consistent with the inventors'prior tissue findings in P301L mice following intracarotid injection ofcontrol IgG (Asuni, A. A. et al. (2007) “Immunotherapy TargetingPathological Tau Conformers In A Tangle Mouse Model Reduces BrainPathology With Associated Functional Improvements,” J. Neurosci.27:9115-9129). Wild-type (Wt) or mice with Aβ plaques (Tg-SwDI, (Davis,J. et al. (2004) “Early-Onset And Robust Cerebral MicrovascularAccumulation Of Amyloid Beta-Protein In Transgenic Mice Expressing LowLevels Of A Vasculotropic Dutch/Iowa Mutant Form Of Amyloid Beta-ProteinPrecursor,” J. Biol. Chem. 279:20296-20306) had minimal brain signal atall time points, highlighting the specificity of the probes for Taupathology. As with the i.c. injection, the scFv or Tau antibodyco-localized with pathological Tau and the probes were detected in theendosome-autophagosome-lysosome system within neurons (FIGS. 3A-3D,FIGS. 4A-4D), and to some extent in microglia.

scFv235 was found to co-localize with pathological Tau within brainneurons following intracarotid injection in P301L (F6) and htau/PS1(A47) tangle mice (FIG. 5). Brains were harvested after the last imagingsession. Tau aggregates were detected with PHF-1 and Tau-5 antibodies,and show partial co-localization with scFv235. A nuclear-specific stainwas used to identify the cell nuclei. FIG. 5 also shows that controlwild-type mice (R1) did not have detectable brain uptake of scFv235after intracarotid injection. Tau-5 stained mainly normal axonal Tau andpathological PHF1 staining was not observed.

In sum, a comparison of the degree of the IVIS signal, residual scFv orantibody signal from sectioned brains and the extent of Tau pathology,after both routes of administration, revealed excellent correlationbetween these parameters (FIGS. 4A-4D, Table 7, Table 8 (Parts A-C),FIG. 5 and FIGS. 6A-6F), scFv: IVIS signal vs. brain tissue scFv signal,r=0.93, p<0.0006; IVIS signal vs. brain Tau pathology, r=0.86, p<0.0002;brain tissue scFv signal vs. brain Tau pathology, r=0.87, p<0.01;6B2G12: IVIS signal vs. brain tissue 6B2G12 signal, r=0.87, p=0.1; IVISsignal vs. brain Tau pathology, r=0.9, p<0.0004; brain tissue 6B2G12signal vs. brain Tau pathology, r=0.8, p=0.05. These findings indicatethat this particular scFv, the parental antibody, and the overallapproach worked very well to detect and assess the degree of Taupathology in live animals.

TABLE 7 Overview of IVIS Imaging Studies of Mice That ReceivedIntracarotid or Intravenous Injection with Diagnostic Probes scFv235 or6B2G12 Antibody Injection Route and Probe Mice Results Objective: UptakeOf scFV235 In The Brain And Binding To Pathological Tau Within NeuronsConjugated Dye: Alexa Fluor 568 Intracarotid - 3 htau/PS1 Good scFvsignal that co- localizes well with Tau5 and MC1 antibody. scFv235Intracarotid - 2 WT Limited scFv signal scFv235 Objective: Live IVISimaging for scFv235/6B2G12 Conjugated Dye: VIVOTAG 680 XL FLUOROCHROME ™IVIS Signals Staining Signal Intracarotid - 3 htau/PS1 Imaging notperformed for Strong scFv signal that co-localizes well scFv235 B32mouse with Tau aggregates and endosome/autophagosome/lysosome (e/a/l)markers for B32 mouse Weak signal for BB5 mouse Weak scFv signal and noTau pathology for BB5 mouse Strong signal in the right Strong scFvsignal that co-localizes well hemisphere that slowly with Tau aggregatesand e/a/l markers for decreases up to 4 h for A47 A47 mouse mouseIntracarotid - 4 P301L Strong scFv signal that is Strong scFv signalthat co-localizes well scFv235 sustained even after 5 h with Tauaggregates and e/a/l markers Intracarotid - 2 WT and 1 Weak signal WeakscFv signal. Limited (htau) or no scFv235 htau (wt) Tau pathologyIntracarotid - 2 P301L Modest signal Weak signal that does notco-localize with Free Dye Tau aggregates Intracarotid - 3 P301L Strongsignal that slowly Strong 6B2G12 signal that co-localizes 6B2G12decreases the next 3-5 h well with Tau aggregates and e/a/l markersIntracarotid - 3 WT Weak signal Weak 6B2G12 signal; No Tau pathology6B2G12 Intravenous - 3 P301L Strong signal up to 1 h that Weak scFvsignal after 7-31 days that co- scFv235 slowly decreases the next 3 hlocalizes well with Tau aggregates and e/a/l markers Intravenous - 1TgSwDl, Weak Signal Weak scFv and Tau antibody signals scFv235 1 WTIntravenous - 3 P301L Strong signal up to 1 h that Weak signal after7-31 days that co- 6B2G12 slowly decreases the next 3 h localizes wellwith Tau aggregates and e/a/l markers Intravenous - 1 Tg-SwDI, Weaksignal Weak 6B2G12 signal. No Tau pathology 6B2G12 1 WT Intravenous - 2P301L Modest signal Weak control IgG signal. Extensive Tau Control IgGpathology, but control IgG does not co- localize with Tau aggregates

TABLE 8 IVIS Signal (Maximum Total Radiant Efficiency) AndSemi-Quantitative Assessment Of Probe Signal From The Brain Sections,Tau Pathology And Co-Localization Of The Injected Probe With VariousMarkers Of The Tau Protein And The Endosome-Autophagosome-LysosomeSystem (Part A) Injected Material/Route IVIS-Maximum Age Intracarotid(IC) Total Radiant Brain Removal Mice ID Strain (Months) Intravenous(IV) Efficiency After Injection BB8 P301L 8 IC- scFv235 1.86E+11 180minutes A12 P301L 12 IC- scFv235 2.64E+11 330 minutes A13 P301L 11 IC-scFv235 2.23E+11 252 minutes F6 P301L 3 IC- scFv235 1.88E+11 280 minutesA50 P301L 9 IC - 6B2G12 1.16E+11 291 minutes A52 P301L 9 IC - 6B2G121.23E+11 235 minutes C1 P301L 7 IC - 6B2G12 2.20E+11 Day 7  B32 htau/PS123 IC - scFv235 — 120 minutes A47 htau/PS1 22 IC- scFv235 1.40E+11 200minutes BB5 htau/PS1 7 IC- scFv235 2.56E+10 200 minutes R1 WT 11 IC-scFv235 4.36E+10 125 minutes 4 WT 8 IC- scFv235 1.34E+10 265 minutes N32htau 11 IC- scFv235 3.87E+10 Day 7  A56 htau/PS1 23 IC - 680 Dye7.45E+10 122 minutes D24 P301L 7 IC - 680 Dye 6.56E+10 165 minutes 1 WT8 IC - 6B2G12 1.22E+10 290 minutes 2 WT 8 IC - 6B2G12 1.48E+10 225minutes 3 WT 8 IC - 6B2G12 1.21E+10 180 minutes A55 P301L 9 IV- scFv2352.07E+11 Day 7  B11 P301L 13 IV- scFv235 2.28E+11 Day 31 E19 P301L 8 IV-scFv235 1.13E+11 Day 7  A49 P301L 11 IV - 6B2G12 1.48E+11 Day 12 C2P301L 7 IV - 6B2G12 1.85E+11 Day 12 B15 P301L 13 IV - 6B2G12 7.62E+10Day 31 VN139 Tg-SwDI 12 IV- scFv235 1.58E+10 Day 31 6 WT 8 IV- scFv2351.11E+10 Day 7  VN142A Tg-SwDI 12 IV - 6B2G12 1.49E+10 Day 31 7 WT 8IV - 6B2G12 8.95E+09 Day 7  D13 P301L 13 IV- Control IgG 8.94E+10 Day 16D15 P301L 13 IV- Control IgG 9.03E+10 Day 16 (Part B) Brain Signal InBrain Tau Co-Localization With Tau Markers Mice ID Sections PathologyTau5 MC1 PHF1 BB8 + + + + + + + + + + + + + + +A12 + + + + + + + + + + + + + + + + +A13 + + + + + + + + + + + + + + + + F6 + + + + + + + + + + + + + +A50 + + + + + + + + + + + + + + A52 + + + + + + + + + + + + + + +C1 + + + + + + + + + + + + + + + B32 + + + + + + + + + + + + + + +A47 + + + + + + + + + + + + + + + BB5 + 0 0 0 0 R1 + 0 0 0 0 4 + 0 0 0 0N32 + 0 + 0 + A56 + + + 0 0 0 D24 ++ + + + 0 0 0 1 + 0 0 0 0 2 + 0 0 0 03 + 0 0 0 0 A55 + + + + + + + + + + + + + + + +B11 + + + + + + + + + + + + + + + E19 + + + + + + + + + + + + + + +A49 + + + + + + + + + + + + + + + C2 + + + + + + + + + + + + + + +B15 + + + + + + + + + + + + + + VN139 0 0 0 0 0 6 + 0 0 0 0 VN142A 0 0 00 0 7 + 0 0 0 0 D13 + + + + + 0 0 0 D15 + + + + + 0 0 0 (Part C)Co-Localization With Endosome/Lysozyme/Autophagy Markers Mice ID EEA1Rab7 LC3 P62 BB8 + + + + + + + + + + + + A12 + + + + + + + + + + + +A13 + + + + + + + + + + + + + F6 + + + + + + + + + + + + +A50 + + + + + + + + + + + + A52 + + + + + + + + + + + +C1 + + + + + + + + + + + + B32 + + + + + + + + + + + + +A47 + + + + + + + + + + + + + BB5 0 0 0 0 R1 0 0 0 0 4 0 0 0 0 N32 + 0 00 A56 0 0 0 0 D24 0 0 0 0 1 0 0 0 0 2 0 0 0 0 3 0 0 0 0A55 + + + + + + + + + + + + B11 + + + + + + + + + + + +E19 + + + + + + + + + + + + A49 + + + + + + + + + + + +C2 + + + + + + + + + + + + B15 + + + + + + + + + + + + VN139 0 0 0 0 6 00 0 0 VN142A 0 0 0 0 7 0 0 0 0 D13 − − − − D15 − − − −

Thus, in summary, scFv molecules of a monoclonal Tau antibody weregenerated using phage display technology. Following extensivecharacterization, scFv235 was assessed as a diagnostic imaging probe inlive tauopathy mice. scFv235 consistently showed strong brain signalafter peripheral injection in two tauopathy tangle mouse models but notin wild-type or Aβ plaque mice. Administration of the parent antibodyled to similar results, but at a lesser signal intensity. Both probesco-localized with intraneuronal Tau aggregates and markers of theendosome/autophagosome/lysosome pathways.

The data show that Tau antibodies and their derivatives administeredperipherally can be used to image Tau brain lesions in live animals.Importantly, the brain signal intensity correlated very well with theextent of the Tau pathology with major implications for experimental andclinical use of this approach. It can be used in this form fornon-invasive monitoring of Tau pathology and treatment efficacy inanimal models of tauopathy, and such ligands have great potential asclinical PET ligands for the same purpose.

The excellent correlation between the IVIS signal, residual probe signalin the brain tissue and the Tau pathology confirms that this approach isuseful for the diagnosis of Alzheimer's disease and other tauopathy inhumans and related animal models and that it provides a means formonitoring treatment efficacy in such patients and related animalmodels.

The scFv molecule or its parent antibody were detected primarily inneurons, co-localized with pathological Tau proteins in theendosome-autophagosome-lysosome system. This is consistent with thesimilar distribution of labeled Tau antibodies and their Fab fragmentsthat is observed after the intracarotid injection or administration ofsuch molecules to brain slice cultures derived from tauopathy mice (Gu,J. et al. (2013) “Two Novel Tau Antibodies Targeting The 396/404 RegionAre Primarily Taken Up By Neurons And Reduce Tau Protein Pathology,” J.Biol Chem. 288:33081-33095; Congdon, E. E. et al. (2013) “AntibodyUptake into Neurons Occurs Primarily via Clathrin-dependent FcgammaReceptor Endocytosis and Is a Prerequisite for Acute Tau ProteinClearance,” J. Biol Chem. 288:35452-35465; Asuni, A. A. et al. (2007)“Immunotherapy Targeting Pathological Tau Conformers In A Tangle MouseModel Reduces Brain Pathology With Associated Functional Improvements,”J. Neurosci. 27: 9115-9129; Krishnamurthy, P. K. et al. (2011)“Mechanistic Studies Of Antibody Mediated Clearance Of Tau AggregatesUsing An Ex Vivo Brain Slice Model,” Front. Psychiatry 2:59). However,this is the first report detecting such aggregates in vivo usingantibodies or their fragments as imaging agents. Prior preclinical andclinical studies targeting α-synuclein or the Tau protein demonstratethat antibodies have substantial access into brains with aggregates ofthese peptides/proteins, presumably because of associated inflammationthat leads to opening of the blood-brain barrier (Congdon. E. E. et al.(2014) “Harnessing The Immune System For Treatment And Detection Of TauPathology,” J. Alzheimers. Dis. 40:S113-S121; Lemere, C. A. et al.(2010) “Can Alzheimer Disease Be Prevented By Amyloid-BetaImmunotherapy?,” Nat. Rev. Neurol. 6:108-119).

There are several possible explanations for the lower signal of theantibody compared to the scFv. The antibody is about 6 times the size ofthe scFv (150 kDa vs. 25 kDa) which may result in less brain andneuronal uptake. The injections are both of the same weight (250 μg),and thus contain 6 times fewer molecules of the antibody compared to thefragment. This initial dose was chosen based on a previous study ofbrain uptake of tagged polyclonal anti-Tau mouse IgG (Asuni, A. A. etal. (2007) “Immunotherapy Targeting Pathological Tau Conformers In ATangle Mouse Model Reduces Brain Pathology With Associated FunctionalImprovements,” J. Neurosci. 27:9115-9129). The higher affinity of theantibody for Tau, compared to the scFv may counteract these issues.

The weak IVIS brain signal in Tg mice with limited Tau pathology,further supports the conclusion that the enhanced signal in the Tg miceis not a mere reflection of Tau overexpression but rather indicates thepresence of Tau pathology. JNPL3 (P301L) mice exhibit stronger brain andperipheral signal than htau/PS1 mice. Within the brain, this fits withmore severe Tau pathology. The greater peripheral signal observed inP301L mice appears in part to be derived from the spinal cord which isknown to have extensive Tau lesions in this model (Lewis, J. et al.“Neurofibrillary Tangles, Amyotrophy And Progressive Motor DisturbanceIn Mice Expressing Mutant (P301L) Tau Protein,” Nat. Genet. 25,402-405). Also, the prion promoter of its mutated Tau transgene resultsin more global Tau expression than in the htau/PS1 model. This may leadto Tau aggregation in various organs.

For animal studies, IVIS imaging is more cost-effective than positronemission tomography (PET) studies and involves a much simpler probepreparation; its labeling with a near infrared dye instead of morecomplicated radiolabeling procedure, which may require an on-sitecyclotron. Hence, IVIS imaging is ideal for probe development to selectligands for subsequent PET studies. Such studies further indicate thatit is an efficient way to monitor the development of Tau pathology andto screen for Tau therapy-mediated clearance of Tau aggregates, witheach animal serving as its own control during longitudinal studies.

The findings strongly support the ability and use of Tau antibodiesand/or their scFv molecules to detect Tau lesions in vivo. In contrastto β-sheet binding dyes, Tau s antibody-derived probes (e.g., scFv235)targeting different epitopes are believed to provide more detailedpicture of the pathological profile of Tau proteins in each individual.This information is envisaged to be able to guide the treatment regimen,which may include active or passive Tau immunotherapy targeting the sameTau epitopes that were detected with imaging. Such a tailored approachis likely to be more efficacious to slow the progression of thetauopathy that is being targeted (see, e.g., Gu, J. et al. (2013) “TwoNovel Tau Antibodies Targeting The 396/404 Region Are Primarily Taken UpBy Neurons And Reduce Tau Protein Pathology,” J. Biol Chem.288:33081-33095; Congdon, E. E. et al. (2013) “Antibody Uptake intoNeurons Occurs Primarily via Clathrin-dependent Fcgamma ReceptorEndocytosis and Is a Prerequisite for Acute Tau Protein Clearance,” J.Biol Chem. 288:35452-35465; Boutajangout, A. et al. (2010)“Immunotherapy Targeting Pathological Tau Prevents Cognitive Decline InA New Tangle Mouse Model,” J. Neurosci. 30:16559-16566; Asuni, A. A. etal. (2007) “Immunotherapy Targeting Pathological Tau Conformers In ATangle Mouse Model Reduces Brain Pathology With Associated FunctionalImprovements,” J. Neurosci. 27:9115-9129; Boutajangout, A. et al. (2011)“Passive Immunization Targeting Pathological Phospho-Tau Protein In AMouse Model Reduces Functional Decline And Clears Tau Aggregates FromThe Brain,” J. Neurochem. 118:658-667; Boimel, M. et al. (2010)“Efficacy And Safety Of Immunization With Phosphorylated Tau AgainstNeurofibrillary Tangles In Mice,” Exp. Neurol. 224, 472-485 (2010);Chai, X. et al. (2011) “Passive Immunization With Anti-Tau Antibodies InTwo Transgenic Models: Reduction Of Tau Pathology And Delay Of DiseaseProgression,” J. Biol Chem. 286:34457-34467 (2011); Bi, A. et al. (2011)“Tau-Targeted Immunization Impedes Progression of NeurofibrillaryHistopathology in Aged P301L Tau Transgenic Mice,” PLoS. One. 6:e26860;d'Abramo, C. et al. (2013) “Tau Passive Immunotherapy in Mutant P301LMice: Antibody Affinity versus Specificity,” PLoS ONE 8:e62402;Troquier, L. et al. (2012)“Targeting Phospho-Ser422 By Active TauImmunotherapy In The THY-Tau22 Mouse Model: A Suitable TherapeuticApproach,” Curr. Alzheimer Res. 9, 397-405; Kfoury, N. et al. (2012)“Trans-cellular Propagation of Tau Aggregation by Fibrillar Species,” J.Biol. Chem. 287:19440-19451; Theunis, C. et al. (2013) “Efficacy AndSafety Of A Liposome-Based Vaccine Against Protein Tau, Assessed InTau.P301L Mice That Model Tauopathy,” PLoS. One. 8:e72301: Yanamandra,K. et al. (2013) “Anti-Tau Antibodies That Block Tau Aggregate SeedingIn Vitro Markedly Decrease Pathology And Improve Cognition in vivo,”Neuron 80:402-414; Castillo-Carranza, D. L. et al. (2014) “SpecificTargeting Of Tau Oligomers In Htau Mice Prevents Cognitive ImpairmentAnd Tau Toxicity Following Injection With Brain Derived Tau OligomericSeeds,” J. Alzheimers. Dis. 40:S97-S111; Castillo-Carranza, D. L. et al.(2014) “Passive Immunization with Tau Oligomer Monoclonal AntibodyReverses Tauopathy Phenotypes without Affecting HyperphosphorylatedNeurofibrillary Tangles,” J. Neurosci. 34:4260-4272). The availabilityof selective biomarkers to determine target engagement, clearance anddisease progression will be essential to avoid the lengthy, expensiveand inconclusive studies in previous Alzheimer's clinical immunotherapytrials. Imaging of these Tau lesions will be very helpful to evaluatethe efficacy of future trials as well as to diagnose Aβ negativetauopathies.

Example 4 Specific and Epitope Dependent Detection of Tau Aggregates inTransgenic Tauopathy Mice In Vivo

As indicated above, the peripheral injection of a single-chain variableantibody fragment (scFv) generated from antibody 6B2G12 resulted in astrong in vivo brain signal in transgenic tauopathy mice using an InVivo Imaging System (IVIS) but not in wild-type or amyloid-β plaque mice(Krishnaswamy S. et al. (2014) “Antibody Derived in vivo Imaging of TauPathology,” J. Neurosci. 34:16835-16850; hereby incorporated byreference herein). Similar specificity but a weaker signal was observedwith the parent 6B2G12 antibody. Importantly, the intensity of theimaging signal correlated well with the degree of tau pathology and theco-localization of the probe with intraneuronal tau aggregates. Bothattributes were associated with markers of endosomes, autophagosomes andlysosomes, indicating their interaction in these degradation pathways.

A comparison of the signal intensity of various tau antibodies againstdifferent tau epitopes in the same transgenic tauopathy mice (P301L,htau) was conducted by administering single intravenous injections ofdifferent tau antibodies. Two different doses were compared (50 μg vs.250 μg) sometimes in the same animals. Injections were separated by atleast a week interval in order to allow the brain signal to subside tobaseline background values. As expected, the larger dose gave aseveral-fold higher brain signal for all the antibodies, suggesting thattheir brain uptake is not saturated at the lower dose. Generally,administration of the 6B2G12 antibody (nM to pM affinity) led to anapproximately 2-3 fold stronger signal than: (1) the signal provided bya lower affinity (nM to μM), but more phospho-selective antibody thatimmunospecifically binds to the same p396,404 epitope; (2) the signalprovided by a high affinity (nM) antibody that immunospecifically bindsto an epitope that is distinct from the p396,404 epitope; or (3) thesignal provided by a low affinity antibody that immunospecifically bindsto a conformational tau epitope. These differences likely reflect boththe relative prominence of these tau antibody epitopes in these animalsand the affinities of the antibodies. All the antibodies evaluatedshowed specificity for tau pathology, resulting in a much stronger brainsignal in tauopathy mice compared to a very weak brain signal in Aβplaque- (Tg-SwDI) or wild-type mice. Limited signal was also detectedwith control IgG antibody. Importantly, the observed signal correlatedwell with the degree of tau pathology and the intravenously injectedantibodies partially co-localized with stained neuronal tau aggregates(PHF1, MC1 and tau-5) and markers of endosomes, autophagosomes andlysosomes (EEA1, LC3, P62 and Rab7) as has been observed with the 6B2G12antibody.

Overall, these findings indicate that tau antibodies against differentepitopes can be used to assess epitope prominence in vivo in mousemodels. Smaller antibody fragments of such imaging probes with evenbetter brain penetration resulting in a stronger brain signal (such asscFv235) have great potential as clinical imaging ligands.

All publications and patents mentioned in this specification are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference in its entirety. While theinvention has been described in connection with specific embodimentsthereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth.

What is claimed is:
 1. A nucleic acid molecule that encodes: (A) anepitope-binding portion of a Light Chain of a humanized antibody that iscapable of immunospecifically binding to phosphorylated Tau with greaterselectivity than to non-phosphorylated Tau, and comprises: (1) a LightChain CDR1 having the amino acid sequence of SEQ ID NO:12; (2) a LightChain CDR2 having the amino acid sequence of SEQ ID NO:13; (3) a LightChain CDR3 having the amino acid sequence of SEQ ID NO:14; and/or (B) anepitope-binding portion of a Heavy Chain of a humanized antibody that iscapable of immunospecifically binding to phosphorylated Tau with greaterselectivity than to non-phosphorylated Tau, and comprises: (1) a HeavyChain CDR1 having the amino acid sequence of SEQ ID NO:15; (2) a HeavyChain CDR2 having the amino acid sequence of SEQ ID NO:16; and (3) aHeavy Chain CDR3 having the amino acid sequence of SEQ ID NO:17.
 2. Thenucleic acid molecule of claim 1, wherein said encoded protein comprisessaid epitope-binding portion of said Light Chain of said humanizedantibody.
 3. The nucleic acid molecule of claim 1, wherein said encodedprotein comprises said epitope-binding portion of said Heavy Chain ofsaid humanized antibody.
 4. The nucleic acid molecule of claim 1,wherein said encoded protein comprises both said epitope-binding portionof said Light Chain of said humanized antibody and said epitope-bindingportion of said Heavy Chain of said humanized antibody.
 5. The nucleicacid molecule of claim 4, wherein said encoded protein is capable ofimmunospecifically binding to phosphorylated Tau and to a phosphorylatedTau peptide having the amino acid sequence of SEQ ID NO:7.
 6. Thenucleic acid molecule of claim 4, wherein said encoded protein iscapable of co-localizing with a Tau aggregate.
 7. The nucleic acidmolecule of claim 4, wherein said encoded protein is a single domainantibody fragment, an scFv or a diabody.
 8. The nucleic acid molecule ofclaim 7, wherein said encoded protein is an scFv.
 9. The nucleic acidmolecule of claim 8, wherein said encoded protein is an scFv thatcomprises an amino acid sequence selected from the group consisting of:SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21.10. The nucleic acid molecule of claim 4, wherein said encoded proteinis capable of treating Alzheimer's disease in a human subject.
 11. Thenucleic acid molecule of claim 7, wherein said encoded protein iscapable of treating Alzheimer's disease in a human subject.
 12. Thenucleic acid molecule of claim 9, wherein said encoded protein iscapable of treating Alzheimer's disease in a human subject.
 13. Thenucleic acid molecule of claim 4, wherein said encoded protein iscapable of treating a tauopathy in a human subject, wherein saidtauopathy is selected from the group consisting of: frontotemporaldementia, parkinsonism linked to chromosome 17 (FTDP-17), progressivesupranuclear palsy, corticobasal degeneration, Pick's disease,progressive subcortical gliosis, tangle only dementia, diffuseneurofibrillary tangles with calcification, argyrophilic grain dementia,amyotrophic lateral sclerosis parkinsonism-dementia complex, dementiapugilistica, Down syndrome, Gerstmann-Straussler-Scheinker disease,Hallerworden-Spatz disease, inclusion body myositis, Creutzfeld-Jakobdisease, multiple system atropy, Niemann-Pick disease type C, prionprotein cerebral amyloid angiopathy, subacute sclerosingpanencephalitis, myotonic dystrophy, non-guanamian motor neuron diseasewith neurofibrillary tangles, postencephalitic parkinsonism, acutetraumatic brain injury and chronic traumatic encephalopathy.
 14. Thenucleic acid molecule of claim 7, wherein said encoded protein iscapable of treating a tauopathy in a human subject, wherein saidtauopathy is selected from the group consisting of: frontotemporaldementia, parkinsonism linked to chromosome 17 (FTDP-17), progressivesupranuclear palsy, corticobasal degeneration, Pick's disease,progressive subcortical gliosis, tangle only dementia, diffuseneurofibrillary tangles with calcification, argyrophilic grain dementia,amyotrophic lateral sclerosis parkinsonism-dementia complex, dementiapugilistica, Down syndrome, Gerstmann-Straussler-Scheinker disease,Hallerworden-Spatz disease, inclusion body myositis, Creutzfeld-Jakobdisease, multiple system atropy, Niemann-Pick disease type C, prionprotein cerebral amyloid angiopathy, subacute sclerosingpanencephalitis, myotonic dystrophy, non-guanamian motor neuron diseasewith neurofibrillary tangles, postencephalitic parkinsonism, acutetraumatic brain injury and chronic traumatic encephalopathy.
 15. Thenucleic acid molecule of claim 9, wherein said encoded protein iscapable of treating a tauopathy in a human subject, wherein saidtauopathy is selected from the group consisting of: frontotemporaldementia, parkinsonism linked to chromosome 17 (FTDP-17), progressivesupranuclear palsy, corticobasal degeneration, Pick's disease,progressive subcortical gliosis, tangle only dementia, diffuseneurofibrillary tangles with calcification, argyrophilic grain dementia,amyotrophic lateral sclerosis parkinsonism-dementia complex, dementiapugilistica, Down syndrome, Gerstmann-Straussler-Scheinker disease,Hallerworden-Spatz disease, inclusion body myositis, Creutzfeld-Jakobdisease, multiple system atropy, Niemann-Pick disease type C, prionprotein cerebral amyloid angiopathy, subacute sclerosingpanencephalitis, myotonic dystrophy, non-guanamian motor neuron diseasewith neurofibrillary tangles, postencephalitic parkinsonism, acutetraumatic brain injury and chronic traumatic encephalopathy.